Indian River Research and Education Center, Florida ...

Indian River Research and Education Center, Florida University, USA.

School of Agriculture, Ningxia University, China

I

Contents

Identification, Expression, and Functional Analysis of the Galactose metabolism gene

LBGALA and Gene Family in goji berry (Lycium barbarum L.) .................................. 1

Transcriptomic analysis of periwinkle (Catharanthus roseus) in response to Clas

(Candidatus Liberibacter asiaticus) infection and characterization of

infection-promoting effectors ....................................................................................... 3

Low Concentration of Sodium Chloride by Foliar Spraying Promote the Growth and

Photosynthesis of Non-heading Chinese Cabbage ....................................................... 5

Comprehensive Evaluation of Different Fertilizer Schemes on the Growth of

Watermelon Seedlings................................................................................................... 7

Responses of Ziziphus jujuba Mill.cv.Lingwuchangzao Fruit Coloration and

Anthocyanin Synthesis Related Gene Expression to Elevated Temperature and

Drought Stress ............................................................................................................... 8

Screening , Identification and Optimization of Fermentation Conditions of Biocontrol

Strain WQ-6 to Melon Fusarium Wilt Disease ........................................................... 10

Morphology and Glucosinolate Metabolism of Chinese Kale in Response to

Supplementary Far-red Light ...................................................................................... 13

Effects of Zeolitic Urea on Growth and Yield of Wheat ............................................ 16

Changes in soil organic carbon pools and enhanced crop productivity in sugarcane

bagasse-biochar amended Soil under wheat-maize cropping system of Pakistan ...... 17

Nanotechnology for Reduction and Remediation of Nonpoint Source of Pollution .. 19

Compost Utilization in Horticultural Cropping Systems ............................................ 20

Evaluation of nitrate leaching and ammonia volatilization from carbon based

designers’ fertilizers in alkaline calcareous soils ........................................................ 21

Analysis in Vitro Culture and Light Quality Effect of Lewisia cotyledon ................. 22

Nutrient Use Efficiency in Crops: A Comprehensive Approach................................. 24

Proliferation and Flowering in vitro of Tissue Culture Seedlings of Lewisia cotyledon

based on Orthogonal Design ....................................................................................... 26

II

Plant growth regulators and EDTA improve phytoremediation potential and

antioxidant response of Chenopodium ambrosioides L. in a Cd-spiked soil .............. 28

Plants buffer against high light stress-induced transcript of CsGA2ox8 via alternative

splicing to fine-tune gibberellins level and maintain hypocotyl elongation ............... 30

Seeds priming technology as a key strategy to increase crops production under

adverse environmental conditions ............................................................................... 32

Effects of greenhouse new/waste rockwool and nutrient on growth and quality of

pakchoi ........................................................................................................................ 33

Purification ability from constructed wetlands treating sewage treatment plants

effluent and microbial community succession in epiphytic biofilms ......................... 34

Impacts of water flow and suspended sediment on microbial community in epiphytic

biofilm on two submerged macrophytes ..................................................................... 36

Aeration enhanced nutrient removal and increased microbial diversity during

Hydrilla verticillata decomposition in wetlands in winter ......................................... 38

Detection of chlorophyll content in tomato leaves based on portable hyperspectral

imaging technique ....................................................................................................... 40

Research of Volatile Components on Champagne and Japanese Peppermint Stem and

Leaf Tissues ................................................................................................................ 42

Study on Visualization Distribution of Chlorophyll in Tomato Leaves under brackish

irrigation Based on Hyperspectral Imaging Technology ............................................ 44

Comparison and Screening of Total RNA Extraction Methods from Fennel Leaves 46

Analysis of Flavor Compounds in Grapefruit Mint Leaf Stem Based on GC-IMS.... 48

Comparative Analysis of Volatile Components in Leave and Stem of the Mentha

spicala by GC-IMS ..................................................................................................... 50

The Identity of Pathongen Causing Tomato Powdery Mildew in Yinchuan .............. 52

Applications of Environmental Economic Policies in the Prevention and Control of

Agricultural Non-point Source Pollution in China ..................................................... 54

1

Identification, Expression, and Functional Analysis of the Galactose

metabolism gene LBGALA and Gene Family in goji berry

(Lycium barbarum L.)

Yaping Ma1,2, Yun Xie2, Naigong Wang3, Bing Cao2,

Lihua Song2, Handong Gao1

1 College of Forestry, Nanjing Forestry University, Nanjing, China 2 School of Agriculture, Ningxia University, Yinchuan, China 3 Zhongke Redflag Information Technology Co., Ltd., Beijing, China

Objectives Exposing goji berry to elevated CO2 for long periods has been shown to

reduce sugar and secondary metabolite contents, and gene LBGALA (Lycium

barbarum alpha-galactosidase) has been found to play a key regulatory role in sugar

metabolism pathways. The objective of this study was to identify gene LBGALA

family members based on the transcriptome, analyze their character and expression

patterns, further understand the initial regulatory functions to sugar metabolism.

Methods The experiment was conducted in open-top chambers (OTC) with two

treatments: ambient (400 μmol mol−1 CO2), elevated (700 μmol mol−1 CO2), and after

90 and 120 days the root, stem, leaf, and fruit tissue samples were collected for

qRT-PCR expression analysis and rapid amplification of cDNA ends (RACE).

Potential members of the LBGALA gene family were identified based on the de novo

transcriptome database with the Hidden Markov model (HMM) and BLAST

homology searches. Green fluorescent protein (GFP) was used for subcellular

localization assay.

Results Six genes (named LBGALA1~LBGALA6) were identified as members of the

LBGALA gene family, one of which exhibited complete open reading frames (ORFs).

The full-length cDNA fragments of LBGALA (GenBank accession No. MH025913)

obtained by RACE-PCR was 1233 bp encoding 410 amino acids. Bioinformatics

analysis showed that the length of LBGAL family members proteins ranged from 101

to 634 aa, with the predicted molecular weight varied from 11.50 to 71.46 KD and

the isoelectric point in the range to 4.86 to 8.74. Phylogenetic analysis revealed that

LBGALA family members are grouped. All LBGALAs proteins contain the typical

Melibiase domain and conservative motif analysis shows that there are 10 motifs in

LBGALA family proteins. The expression of LBGALAs was expressed in different

2

tissues of goji berry under different CO2 treatments with 90 and 120 days.

Sub-cellular localization showed that LBGALA and LBGALA1 were located in the

chloroplast.

Conclusions Six LBGALA genes from the Lycium barbarum transcriptome were

identified and contain a typical Melibiase domain. The expression patterns of the

LBGALAs under different CO2 concentrations and tissues suggested that significantly

expressed in root, leaf, stem, and fruit. This study provides a basis for further

research on the regulation function of LBGALA to sugar metabolism.

Acknowledgments This work was supported by the National Natural Science

Foundation of China (Grants No. 31660199).

3

Transcriptomic analysis of periwinkle (Catharanthus roseus) in

response to Clas (Candidatus Liberibacter asiaticus) infection

and characterization of infection-promoting effectors

Qiaolin Zheng1, Marco Pitino1, YongPing Duan2

and Liliana M. Cano1

1 University of Florida, Institute of Food and Agricultural Sciences IFAS, Department of Plant

Pathology, Indian River Research and Education Center, Fort Pierce, FL 34945, USA 2 U.S. Horticultural Research Laboratory, USDA-ARS, Fort Pierce, FL 34945, FL, USA

Background Huanglongbing (HLB), the most destructive disease of citrus, causes

quick decline of citrus production all over the world and has rendered millions of

others useless for production. HLB-associated Candidatus Liberibacter asiaticus

(Clas) is the most widespread pathogen in citrus and transmitted by insect vector

Asian citrus psyllid (ACP, Diaphorina citri). HLB was first discovered in the United

States in Florida in 2005, which reduced the value of Florida citrus output by $7

billion during the years from 2005 to 2014 and caused 72.2% reduction in the

production of oranges for processing and 20.5% reduction in the fresh fruit market in

the United States from 2007-08 to 2017-18. In order to develop novel strategies to

completely eliminate HLB in citrus, one of the most important aspects is to clarify

the mechanism of citrus-Clas interactions. Periwinkle (Catharanthus roseus) is

HLB-susceptible host plant and presents similar infected-symptoms observed in

citrus. Moreover, it is easily grown and quickly infected by Clas through grafting.

Clas bacteria present in periwinkle plants can grow faster and reach higher titer level

than in citrus.

Methods Two-month old healthy periwinkle plants were grafted inoculation with

Clas infected branches. The leaves of the grafted branches were collected at 2 and 4

weeks after grafting (wag) for RNA extraction. The expression of a total of 27 Clas

effectors was assayed with RT-qPCR methods in Clas-infected periwinkle leaves.The

transcriptome analysis of periwinkle infected with Clas was performed as well.

Results A total of 14,062 genes were considered differentially expressed using a fold

change cutoff of Gfold (0.01) less than -1 and greater than 1 in Clas-infected

periwinkle at 2 wag, and 9,509 DEGs (Differentially expressed genes) were

identified at 4 wag. Eleven DEGs were validated via qRT-PCR, and all of them were

up-regulated in response to Clas infection in periwinkle, including DEGs involved in

4

transcription factors, secondary metabolism pathways, signal pathways, stress, cell

wall modification, transporters, and starch synthesis. Gene expression of 19 putative

effectors was detectable in Clas-infected periwinkle leaves at early infection stage, and

effector ‘0470’showed the highest expression level. In total, 27 putative effectors were

highly expressed at 4 wag in Clas-infected periwinkle, and the expression level of

each effector was thousands of times higher than that expressed at 2 wag.

Conclusions Transcriptional profiling of the genes involved in Clas infection was

investigated in Clas-infected periwinkle at 2 and 4 wag. Our study indicated larger

numbers of genes with much higher expression level were induced at 2 wag than 4 wag

in response to Clas infection, as well as the up-regulated genes much more than the

down-regulated ones. The numbers of detectable effectors and the transcriptional level

of effector were related to the titers of bacteria in infected periwinkle leaves. The

expression of 19 and 27 effectors were observed at 2 and 4 wag, respectively. The

expression level of effectors at 4 wag was thousands of times higher than that at 2 wag.

5

Low Concentration of Sodium Chloride by Foliar Spraying Promote

the Growth and Photosynthesis of Non-heading Chinese Cabbage

Wei Zhang1, Shuang Gao2, Junlin Wang3, Mei Wang4,

Zhikui Gao1*, Zhanjun Xue1*

1 Collage of Horticulture，Hebei Agricultural University, Baoding , China 2 College of Food Science and Technology, Agricultural University of Hebei, Baoding,China 3 College of Life Sciences, Hebei Agricultural University, Baoding , China 4 Center of Science and Education Service，Hebei Agricultural University, Baoding, China

Objectives Many researches have been paied more attentions for the stress effects of

higher concentration (50-300 mmol·L-1) sodium chloride (NaCl), which were directly

used into soil or nutrient solution. Recently, studies have shown that there are

beneficial effects from the addition of low NaCl as a nutrient solution to the

rhizosphere and this practice promotes the growth of various plants. Although low

concentration NaCl is applied repeatedly in soil cultivation, it is likely to also create

hidden dangers of secondary soil salinization. So the foliar spraying method was used

to study the regulatory effect of low concentration of NaCl on the growth and

photosynthetic activity of non-heading Chinese cabbage and provide a basis for

broadening the application of NaCl.

Methods The non-heading Chinese Cabbage variety 'Hanyue F1' in the three–leaf

stage was used as the test material growing in nutritive bowl under the greenhouse.

Low concentrations of NaCl (6 mmol·L-1) were applied once a day as a foliar spray at

every morning during the growth period with distilled water (0 mmol·L-1) as the

control. After the leaves were sprayed with the low concentrations of NaCl lasting for

18 days, the growth, photosynthesis, water holding and nutrient patameters were

determined.

Results The fresh biomass, dry biomass and water content of the whole plant

increased significantly by 49.5%, 28.7%, and 51.5%, respectively. The water holding

capacity of the WHCplant, leaf area, leaf succulence, the photosynthetic carbon

absorption rate(Pn) and the stomatal conductance Gs also increased significantly.

Simultaneously, treatment with NaCl not only increased the dry biomass of leaves,

particularly the petioles, but also increased the water content of all the organs,

particularly the stem. In addition, the treatment increased the dry biomass water

holding capacity of the leaves and stems of non-heading Chinese cabbage. The

6

content of free amino acids in the leaves and petioles of Chinese cabbage increased

significantly by 73.4% and 58.8%, respectively, following the application of NaCl to

the leaves. In addition, the NaCl treatment in three–leaf stage stimulated the

expanding leaves (the second and third euphyllas), particularly for the leaves that

were about to unfold (the fourth to the eighth euphyllas).

Conclusions Spraying treatment of a low concentration of NaCl not only increased

the fresh content of non-heading Chinese cabbage through the accumulation of water

and dry biomass, but also increased the expansion of leaf area, the light interception

area and the stomatal conductance (GS), so as to improve the Pn and dry biomass

accumulation.

Acknowledgments This work was supported by the Key R & D Projects of Hebei

Province (18226928D).

E-mail to corresponding author [email protected]; [email protected]

7

Comprehensive Evaluation of Different Fertilizer Schemes

on the Growth of Watermelon Seedlings

LIANG Huan, ZHU Juhong, GE Mihong, WANG Dehuan,

ZHOU Mobing, SHI Xianfeng *

Wuhan Academy of Agricultural Sciences，Wuhan 430345

Abstract 【Objective】The effects of different fertilization amount and concentration

on the growth and development of watermelon seedlings were studied in order to

identification the fertilization scheme of watermelon seedling.【Method】Taking the

‘zaojia 84-24’ watermelon seedlings as the research objects, the morphological

parameters, such as the plant height, stem diameter and so on, were measured. A

comprehensive evaluation method containing principal component, membership

functions and system clustering was applied to analyze indices in different fertilizer

treatment. 【Result】There were significant differences in the 11 indexes after

different fertilizer treatment, such as plant height, stem thickness, leaf area, fresh

weight and so on. By principal component analysis, 11 indexes were transformed into

3 independent comprehensive indexes. Cluster analysis showed that D5、E5 and E6

treated seedlings had the best quality. 【Conclusion】The fertilization scheme of

watermelon seedling was as follows: the fertilization rate respectively were 250

mg∙L-1, 250~300 mg∙L-1and 250~350 mg∙L-1 at three developmental stage, including

cotyledon extension, the 1st leave stage and 2nd leave stage.

Key words Watermelon, Plug seedling, Fertilization scheme; Comprehensive

evaluation

8

Responses of Ziziphus jujuba Mill.cv.Lingwuchangzao Fruit

Coloration and Anthocyanin Synthesis Related Gene

Expression to Elevated Temperature

and Drought Stress

Wenqian Jiang, Lihua Song

School of Agriculture, Ningxia University, Yinchuan,China

Background Increased temperature and drought are among the most important

factors challenging sustainable agricultural production worldwide. The world average

temperature is likely to increase by 1.5 °C between 2030 and 2052 and 2.3 ± 0.3 °C

by 2076. Along with these increased temperatures, it is also predicted that there will

be changes to precipitation patterns in various regions. Ningxia, located in

northwestern China, is an arid and semi-arid region. In the past 60 years, the

temperature has been rising and the amount of evaporation has also been increasing.

Therefore, the elevated temperature and drought in Ningxia will become more and

more obvious, which will impact the fruit coloration of ‘Lingwuchangzao’ (Ziziphus

jujuba Mill. cv. ‘Lingwuchangzao’), which is one of featured economic forest tree

species.

Methods We applied the air temperature and drought interaction design. The

treatment of drought stress included three levels (D1, soil moisture is 70 - 75 % of

field capacity; D2, soil moisture is 50 - 55% of field capacity and D3, soil moisture is

30 - 35 % of field capacity), whereas the treatment of air temperature included two

levels (T1, natural air temperature; T2, elevated air temperature=T1＋(2 (±0.5) °C).

With 5-year-old grafted seedlings of jujube cultivar ‘Lingwuchangzao’ as the the

experimental objects, the Open-top chambers (OTC) were used to simulate the

elevated temperature environment, solar automatic irrigation control system to

control the soil moisture. By measuring the fruit sugar, acid content, color index, peel

pigment content, anthocyanin synthase activity and gene expression under different

treatments at different stages, to analyze the effects of elevated temperature and

drought stress on fruit coloration and anthocyanin synthesis of ‘Lingwuchangzao’.

Results 1.The elevated temperature significantly increased a* (red-green difference)

(13.68%) and a*/b* (red-green difference/yellow-blue difference) (10.63%), soluble

sugar content (39.92%), the content of flavonoid and carotenoid, the expression of

9

ZjCHS, ZjF3H, ZjDFR, ZjUFGT and ZjLDOX genes. Drought treatments

significantly decreased a*, a*/b*, soluble sugar content, the content of flavonoid and

carotenoid and this genes expression. Elevated temperature significantly increased

the activity of UFGT (6.90%), PAL (6.20%), and DFR (5.47%) during the coloration

stage, while drought treatments significantly reduced the activity of UFGT, PAL, and

DFR, especially the severe drought treatment.

2.The L* and h* of the D3T1 treatment combination were the highest, which

were significantly higher than other treatment combinations, indicating that the fruits

under D3T1 treatment combination had poor coloration. In the full-ripening stage, the

peel anthocyanin content, the activity of UFGT, PAL and CHI, the ZjF3H gene

expression of the D1T2 treatment combination was significantly higher than D1T1

treatment combination.

3. Soluble sugar content was always significantly or highly significantly

positively correlated with anthocyanin content during the coloration and full-ripening

stage (r=0.526*, 0.677**), and the organic acid content is highly significantly

negatively correlated with anthocyanin content during the coloration stage (r=

-0.894**).

4. During the fruit coloration stage and the full-ripening stage, the activity of

UFGT, PAL, and DFR were significantly or highly significantly positively correlated

with the synthesis and accumulation of anthocyanin. And ZjLDOX, ZjUFGT and

ZjDFR are the key genes for the anthocyanin synthesis pathway of ‘Lingwuchangzao’

peel under elevated temperature and drought in the fruit coloration process.

Conclusions The elevated temperature by about 2.0℃ promoted the coloration of

‘Lingwuchangzao’ by increasing the soluble sugar content of fruits, and promoted the

activity of UFGT, PAL, DFR and the expression of ZjCHS, ZjDFR, ZjUFGT, and

ZjLDOX genes. Drought stress is not conducive to fruit coloration. ZjLDOX, ZjUFGT

and ZjDFR are the key genes in the anthocyanin synthesis pathway of the pericarp of

‘Lingwucahngzao’. Elevated temperature with normal soil moisture is the best

environment of ‘Lingwuchangzao’ fruit coloration by promoting UFGT, PAL, CHI

activity and ZjCHS, ZjF3H gene expression.

Acknowledgements This work was supported by the National Nature Science

Foundation of China (Grants No. 31760203).

10

Screening , Identification and Optimization of Fermentation

Conditions of Biocontrol Strain WQ-6 to

Melon Fusarium Wilt Disease

Wenwen Zhang, Qing WU, Cui Yin, and Yune Cao

School of Agriculture,Ningxia University,Yinchuan,China

Background A lot of studies have been done on the screening and application of

antagonistic bacteria against wilt of cucurbit crops. But most of the studies. The

biocontrol bacteria in the plant is only targeted at a single type of fusarium oxysporum,

which has a single effect in disease control and has great limitations in production and

application. It is necessary to screen the biocontrol bacteria that have broad spectrum

resistance to various fusarium oxysporum. Metabolic active substances are the key to

the biocontrol effect of bacterial strains. Appropriate fermentation technology is the

basis for obtaining a large number of metabolically active substances from

microorganisms. Based on this, in this study different cucumber, watermelon and The

fusarium wilt bacteria of muskmelon was selected as the target bacteria, and the

antibacterial properties of the strains and their metabolites were selected as the

screening conditions to be screened from the earthworm compost. The birth control

strain was selected to study its broad-spectrum control effect and application

effect.The antagonistic strains were optimized by single factor test and response

surface analysis. In this paper, the effect of antagonistic strain on melon fusarium wilt

was verified by seed germination test and pot experiment, and the antagonistic strain

was studied. In order to provide new species resources for the biological control of

cucurbit fusarium wilt, the biocontrol effect of plant compound and earthworm

composting extract on fusarium wilt was studied. Field application lays the foundation.

Methods Four kinds of fusarium wilt pathogens (2 kinds of cucumber, 1 kind of

watermelon and 1 kind of melon) were selected as the pathogens, and the flat plate

confrontation method combined with the method of inhibiting mycelium growth rate

was adopted to select 1 strain of antagonistic bacteria from earthworm compost that

had significant inhibitory effect on all four kinds of pathogenic bacteria. Physiological,

biochemical and molecular identification were carried out for the screened strains with

inhibitory effect to determine the species. The optimum fermentation technology was

obtained by using single factor test and response surface analysis method, which

11

increased the antibacterial diameter and antibacterial rate of bacterial strain

fermentation liquid against fusarium wilt. Finally, the antagonistic bacteria were tested

to meet the environmental requirements of biocontrol agents to ensure that they could

colonize the root system of crops.

Results A new strain WQ-6 exhibiting antagonism towards all four pathogens tested，

was screened out using the methods of plate-confrontation and inhibition mycelial

growth and was identified as Bacillus siamensis. Based on single factor test and

response surface analysis，an response surface analysis，an optimal fermentation

condition for strain WQ-6（i.e. glucose 30 g·L -1,peptone 30 g·L-1 ，magnesium sulfate

1g·L-1,glycerol 1g·L-1,dipotassium hydrogen phosphate 1.5g·L-1,temperature 32.2 ℃，

liquid-loading rate 19.2%,rotating speed 236.2 r·min-1，pH 6.0，inoculation rate 4%

and cultural time 24 hours）was obtained. Under the optinal condition，the inhibition

zone diameter increased by 3.52–10.45 mm and the inhibition rate of pathogens

increased by 2.28%–32.16%,as compared to the unoptimized control. The seed

germination test (cucumber，watermelon and muskmelon）and pot experiment showed

that the strain WQ-6 reduced the disease incidence of seeds by 32%–68%，increased

germination rate by 100%–259.6%，and decreased the disease index of plants by 20%–

34%. In addition, the WQ-6 fermentation broth（WFB）mixed with water extract of

vermicompost significantly enhanced the root length，stem length and vigor index of

seeds，and reduced the disease index of plants by 6.78%–36.44%,as compared with

WFB alone.

Conclusions Among the existing biocontrol bacteria against cucurbit fusarium wilt,

most of them are bacteria, especially bacillus. Bacillus has the ability to inhibit a

variety of plant pathogens and is a widely used biocontrol strain. In this study, B.

Siamensis had a good antagonistic effect on cucumber Fusarium wilt, melon Fusarium

wilt and watermelon fusarium wilt (the inhibitory diameter was 28.32 ~ 31.32 mm, and

the inhibitory rate was 51.65% ~ 59.15%).The selection of medium components

suitable for the growth of the strain and the optimum fermentation conditions were

beneficial to increase the fermentation yield of the strain and give full play to its

biocontrol potential. In this study, based on the single factor experiment, response

surface analysis method designed by Plackett Burman experiment Design and Central

Composite Design was adopted to optimize the fermentation process of strain WQ-6 as

a whole, which simplifies the test steps, is intuitive and has high reliability. Compared

12

with that before optimization, the biocontrol ability of the strain was significantly

increased, and the inhibition diameter and inhibition rate of fusarium wilt increased by

14.1% ~ 50.1% and 4.3% ~119.2%, respectively. At the same time, the control effect

test results showed that the incidence of seed decreased by 32% ~ 68%, the

germination rate increased by 100% ~ 259.58%, and the disease index of plant

decreased by 20% ~ 34%.The results showed that wQ-6 had a good effect of

antagonizing cucurbit wilt and promoting growth. The root length, stem length and

vigor index of cucurbit seed germination were significantly higher than that of

pathogen treatment with wQ-6 fermentation broth alone, and the prevention and

control effect on plants was improved by 6.78% ~ 36.44%, indicating that wQ-6

combined with earthworm compost extract could improve its ability to prevent

diseases and promote growth. At the same time, the fermentation conditions of wQ-6

strain were optimized as a whole to determine a set of fermentation mode with the

highest yield, the most economical and effective, which laid a foundation for deep

fermentation of WQ-6 strain and large-scale industrial production, and provided new

biocontrol resources for the prevention and control of soil-borne diseases of facility

vegetables.

13

Morphology and Glucosinolate Metabolism of Chinese Kale

in Response to Supplementary Far-red Light

Yamin Li1 and Houcheng Liu1

1 College of Horticulture, South China Agricultural University, Guangzhou 510642, China

Objectives Chinese kale (Brassica alboglabra Bailey) is one of the most popular

Brassicaceae vegetables in South China, which is known for high nutritional value

and abundant glucosinolates (GSL). GSL are an important group of secondary

metabolites that participate in plant stress responses and flavor formation (pungency

and bitterness). Although far-red light is beyond the visible light, it has been proven

to greatly influence plant growth, development, as well as metabolite accumulation.

This study aims to explore how Chinese kale in response to far-red light.

Methods The Chinese kale cv. ‘Lv Bao’ were cultivated under 250 μmol m–1 s–1

white light-emitting diodes (6:00 - 18:00) in the plant factory. There were three

supplemental far-red (735 nm) light treatments: 0 W/m2 (CK), 3 W/m2 (FR-3) and 6

W/m2 (FR-6). The transcriptomics profiles, agronomic traits, and individual GLs

were combined to investigate the regulation mechanism of Chinese kale in response

to far-red light.

Results (1) Along with the increased far-red light the plant display, plant height,

internode length, leaf length, leaf width, and leaf area of Chinese kale significantly

enlarged, while the leaf angle narrowed. (2) Higher supplemental far-red light

obviously accelerated the transformation from vegetative growth to reproductive

growth in Chinese kale by increasing the flower budding rate at 60 days after sown.

(3) Nine individual GSL were detected in Chinese kale. The total GSL, aliphatic GSL,

and indolic GSL were significantly decreased by far-red light, especially FR-6. Eight

kinds of GLs had the similar decreased trend with total GSL, while the content of

4-Hydroxyglucobrassicin were increased by FR-3. (4) Based on Brassica database,

210 genes were identified as GSL biosynthesis genes homologous to Arabidopsis

thaliana. In the chain elongation step, genes annotated MAM1/2, IPMDH and BAT5

were down-regulated by far-red light. In the core structure formation step, genes

coding CYP79F1, CYP83A1, SUR1, UGT74B1, UGT74C1, GSTU20, and GSTF9/10

were also down-regulated by far-red light.

14

Conclusions In conclude, higher far-red light supplementation showed stronger

influence on the growth and metabolism of Chinese kale. The results indicate that

far-red light supplementation could be used strategically to modify a larger and more

compact appearance of Chinese kale. Meanwhile, far-red light supplementation is

efficient in alleviating the pungency and bitterness flavor by suppressing the GSL

biosynthesis and consequently reducing the GSL contents of Chinese kale.

Acknowledgements This work was supported by Key Research and Development

Program of Guangdong (2019B020214005, 2019B020222003).

Figure 1. Effect of supplementary far-red light on Chinese kale morphology.

The morphological indices (a-k) of Chinese kale 60 days after sown.

15

Figure 2. Effect of supplementary far-red light on the contents of glucosinolates (GSL) in Chinese kale.

Relative abundance of (a) aliphatic and (b) indolic GSL is indicated from blue (low) to orange (high). (c) Relative

expression of genes involved in GSL biosynthesis is indicated from green (low) to red (high). PRO = progoitrin,

RAA=glucoraphanin, SIN=sinigrin, GNA=gluconapin, GBN=glucobrassicanapin, 4OH=4-hydroxyglucobrassicin,

GBC=glucobrassicin, 4ME=4-methoxyglucobrassicin, NEO=neoglucobrassicin.

PRO

RAA

SIN

GNA

GBN

4OH

GBC

4M E

NEO

CKFR-3

FR-6

-1.20

-0.90

-0.60

-0.30

0.00

0.30

0.60

0.90

1.20

M ethionine

RAA GBNSIN

GNA

Aliphatic GSL

PRO

(a)

Desulfo GSL

PRO

RAA

SIN

GNA

GBN

4OH

GBC

4M E

NEO

CK FR-3

FR-6

-1.20

-0.90

-0.60

-0.30

0.00

0.30

0.60

0.90

1.20

PRO

RAA

SIN

GNA

GBN

4OH

GBC

4M E

NEOCK

FR-3

FR-6

-1.20

-0.90

-0.60

-0.30

0.00

0.30

0.60

0.90

1.20

PRO

RAA

SIN

GNA

GBN

4OH

GBC

4M E

NEO

CK

FR-3

FR-6

-1.20

-0.90

-0.60

-0.30

0.00

0.30

0.60

0.90

1.20

PRO

RAA

SIN

GNA

GBN

4OH

GBC

4M E

NEO

CK

FR-3

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16

Effects of Zeolitic Urea on Growth and Yield of Wheat

Ayaz Ahmad1, Shahzada Sohail Ijaz1, Khalid Saif Ullah Khan1,

Muhammad Ansar1, Mahnoor Azad1

1 Institute of Soil Science, PMAS-Arid Agriculture University, Rawalpindi, Pakistan

Objectives Urea is the most commonly used fertilizer in agriculture but its losses are

high (40-70%) due to quick release of Nitrogen. Negatively charged zeolites are

known to act as sorbent of the nutrients. The adsorbed nutrients are released gradually.

Objectives of the study were to test nutrient release pattern of the Zeolite based

fertilizer and its effect on growth and yield of wheat.

Methods Two year field experiment was conducted on medium texture soil.

Treatments were: Control, N @ 114 kg ha-1 from urea, N @ 114 kg ha-1 from zeolite

based fertilizer, N @ 85.5 kg ha-1 from zeolite based fertilizer, N @ 57 kg ha-1 from

zeolite based fertilizer. Soil samples were collected at tillering, booting and maturity

stages of wheat crop and analyzed for total N, NO3- N and NH4-N. Plant samples

were collected at each stage and analyzed for total N. At crop maturity biomass and

grain yields were measured.

Results Results implied that zeolite based fertilizer enhanced yield of wheat crop

even at 75% of N dose as compared with the full dose of N from urea. Plant uptake of

N was also high with zeolitic urea.

Conclusion It is concluded that zeolitic urea has potential to slow release of nitrogen

and to improve nitrogen use efficiency of urea fertilizer.

17

Changes in soil organic carbon pools and enhanced crop productivity in

sugarcane bagasse-biochar amended Soil under wheat-maize

cropping system of Pakistan

Muhammad Irfan*1, Qaiser Hussain1, Khalid Saifullah Khan1,

Azeem Khalid2, Muhammad Azeem3, Sana Ur Rehman4

1 Institute of Soil Science PMAS-Arid Agriculture University Rawalpindi, 46300, Punjab

Pakistan 2 Department of Environmental Sciences PMAS-Arid Agriculture University Rawalpindi,

46300, Punjab Pakistan 3 Department of Environment Science & Engineering College of Natural Resources and

Environment Northwest A&F University, Yangling, Shaanxi, 712100, P. R. China

4 Land Resources Research Institute, National Agriculture Research Center Islamabad

Corresponding Author: Muhammad Irfan (Ph.D Scholar)

Corresponding Author Email: [email protected]

Abstract Intensive agricultural practices results in soil degradation through loss of

soil organic matter. Amending soils with biochar have multiple environmental benefits,

including improvement in soil physicochemical properties, reduction in leaching losses

of essential nutrients and reduction in greenhouse gas (GHG) emissions. The present

study was conducted to evaluate the direct and residual effect of biochar in

combination of N fertilizer on carbon fractions in wheat-maize cropping system of

arid region of Pakistan. Biochar (sugarcane straw) prepared by conventional

pyrolysis, having with seven treatments in experiment B0N0=control (no biochar and

recommended urea fertilizer), B1N0=0.5% biochar C ha-1, B2N0=1% biochar C ha-1,

B0N1= recommended N fertilizer (100 kg ha-1), B1N1=0.5% biochar C ha-1+

recommended urea fertilizer, B2N1=1% biochar C ha-1+ recommended urea fertilizer.

The biochar was applied before the sowing of wheat and further residual effect was

studied on the other crop (maize). The results of the two years field study showed that

biochar amendment increased soil pH, soil organic carbon (SOC), carbon fractions

(dissolved organic carbon, mineralizable organic carbon hot water extractable carbon

and HCl insoluble carbon) and also on crop productivity. However, results also

indicated that water soluble carbon and soil bulk density was gradually decreased

during the both growing cycles. The soil organic carbon (SOC) and labile fractions

significantly enhanced with increasing biochar application dose over the course of the

experiment suggesting that biochar application has great potential for carbon

sequestration in the arid soil. The results therefore also suggest that addition of

18

biochar with recommended urea fertilization could have significant effect on

biological and grain yield of both crops. The outcomes propose that biochar

amendment to carbon deficient soils had an appropriate management practice to

enhance soil carbon accumulation and sequestration. This whole two year field study

was supported by Institute of Soil Science, PMAS-Arid Agriculture University

Rawalpindi, 46300, Punjab, Pakistan.

19

Nanotechnology for Reduction and Remediation of

Nonpoint Source of Pollution

Zhenli He and Xiaoping Xin

University of Florida, Institute of Food and Agricultural Sciences, Department of Soil

and Water Science/Indian River Research and Education Center, Fort Pierce, FL 34945,

United States

Abstract To meet the ever-increasing demand of world population for food and fiber

and remediate nonpoint source of pollution in agriculture, nanotechnology-enabled

agricultural innovations show great promise in delivering a more sustainable,

efficient and resilient agricultural system, while promoting food security and

reducing environmental pollution. Further exploration of nanotechnology

applications in agriculture is necessary to realize its potential in manufacturing

innovative agrochemicals and novel delivery platforms for enhancing crop

production and quality. Here, we reviewed fundamentals of nanotechnology and

focused on its potential in agricultural applications. Progress has been made in the

development of nano-fertilizers, nano-additives, nano-pesticides/herbicides/bactericides,

nano-filters to improve agrochemical efficiency, reduce run-off, enhance plant growth,

and remediate contaminated water and soils. Especially, nano-delivery systems have

been designed to deliver effective components to the targeted sites within a plant to

provide potential solutions to some devastating crop diseases, and carbon

nanoparticles have been used as fertilizer additives to enhance Seed germination,

plant growth and nutrients uptake. However, nano-enabled agriculture is still in its

infancy and its applications are mostly theoretical. Therefore, more research is

needed to develop biodegradable, cost-effective and safe nanomaterials for future

application. Moreover, systematic studies are crucial to safeguard our food

production system, while making efforts to raise public awareness to nanotechnology.

Keywords Agriculture; environmental remediation; nanomaterials; nutrient use

efficiency; soil pollution

20

Compost Utilization in Horticultural Cropping Systems

Peter J. Stoffella

Professor Emeritus, University of Florida, IFAS, Indian River Research and Education

Center, Fort Pierce, Florida, 34951 USA

Biological degradable materials, such as, municipal solid waste, biosolids, yard

trimmings, animal manures, and food processing by-products are being commercially

developed into horticultural grade compost. Commercial compost and be produced

though aerobic (static pile, window, or in-vessel) or anaerobic methods. These

systems produce a viable product for horticultural crop production while

subsequently reduce landfill material as well as extending landfill longevity. Compost

utilization in horticultural crop systems can be a potential alternative to polyethylene

mulch, fumigation, biological weed control in row alleys, source on macro and micro

nutrients, alternative to peat in potted plant culture, and reduce nutrient leaching in

ground water and loads in surface water. Precautions of compost utilization should be

with variability within and between production facilities, maturity issues, heavy

metal content, and availability of application equipment.

21

Evaluation of nitrate leaching and ammonia volatilization from

carbon based designers’ fertilizers in alkaline calcareous soils

Muhammad Rashid1, Qaiser Hussain2*, Khalid Saifullah Khan1,

Rifat Hayat1, and Sarosh Alvi2

1 Institute of Soil Science, PMAS-Arid Agriculture University, Rawalpindi, Pakistan 2 Soil and Water Testing Laboratory, Rawalpindi, Pakistan *Corresponding author: [email protected]

Abstract Chemical fertilizers are considered as costly inputs for adequate crop

production; however, their ever-increasing demand and prices has provoked serious

efforts for the development of nutrient efficient and climate-smart fertilizers. Biochar

and lignite have marked their significance as efficient soil amendments due to their

multiple agricultural and environmental benefits. But, their potential as a fertilizer

nutrient carrier need further research attention. Considering these research gaps

especially under alkaline calcareous soil, slow-release carbon-based designers’

fertilizers were synthesized and the nitrogen losses from these fertilizers in the form

of nitrate leaching and ammonia volatilization were assessed in two different textured

alkaline calcareous soils in comparison with conventional urea and commercial

slow-release fertilizer. Application of slow-release carbon-based designers’ fertilizers

such as Bio-SRF and Lig-SRF reduced the ammonia volatilization by 38.46 and

47.02% in coarse texture soil while, 39.56 and 44.90% in fine textured soil,

respectively, compared with conventional urea. Similarly, reduction of nitrate

leaching due to application these carbon-based designers’ fertilizers was 49.66 and

57.01% in coarse textured soil while 47.64 and 53.32% in fine textured soil,

respectively, compared with conventional urea. The reduction of N volatilization and

leaching losses imparted by carbon-based designers’ fertilizers could be a viable N

source for crops. However, field-level validation experiments could better assess the

efficacy of these carbon-based designers ‘fertilizers in enhancing crop yield and

nutrient use efficiency.

Keywords Slow-release fertilizer; biochar; lignite; ammonia volatilization; nitrate

leaching; alkaline soil

mailto:[email protected]

22

Analysis in Vitro Culture and Light Quality

Effect of Lewisia cotyledon

Tian Tian1, Chun Liu2, Li Zhang1*

1 College of Agriculture, Ningxia University, Yinchuan, 750021; 2 Vegetable and Flower Research Institute, Chinese Academy of Agricultural Sciences,

Beijing, 100086

Background Lewisia cotyledon is a kind of portulaceae flower with thick leaves and

beautiful flowers. It is native to the central mountain area of the west coast in the

United States. Potted plants are often used to decorate restaurants, open bars, private

gardens and rock gardens. 'Elise mix' series is the first hybrid of L. indica. Because of

its several features, such as a long flowering period, the bright color, drought

resistance, heat resistance, and blossoming without low temperature. It is suitable for

potted and open field application .

Methods 1. Callus induction：The young leaves were cut into 0.5 cm× 0.5 cm squares,

and the callus induction medium was inserted into the leaves with the back facing

down. The medium was based on MS, supplemented with sucrose 30 g/L, agar 6 G/L,

6-BA, 2,4-D and NAA.

2. Effects of different light quality and proportion on callus proliferation：The

callus was cut into 0.3 g callus pieces and inoculated into the selected proliferation

medium (MS+30 g/L sucrose+6 g/L agar+2.5 g/L 6-BA+0.1 mg/L NAA, pH=5.8).

After 20 days of culture, the weight gain and the rate of callus weight gain were

counted.In this experiment, eight light quality ratios were set up for LED, which were

100% blue light, 100% red light , 20% blue light + 80% red light , 80% blue light +

20% red light , 30% blue light + 70% red light , 70% blue light + 30% red light , 50%

blue light + 50% red light , PGF (fluorescent lamp for plant tissue culture).

3.Embryogenic callus induction under different light quality：0.3 g calli were

inoculated in the selected callus induction medium, and five kinds of light quality

treatments were set: 100% blue light , 100% red light , 20% blue light + 80% red

light , 70% blue light + 30% red light, PGF . Each treatment was repeated for 10

times, and each bottle was recorded as a repetition. Two calli were grafted into each

bottle. After 30 days of culture, embryogenic callus induction was observed.

Results The results showed that the optimum explants for callus induction were

23

leaves, MS+6-BA 1.0 mg/L+2,4-D 0.2 mg/L+NAA 0.1 mg/L, 20% blue light+80%

red light (B:R=2:8) and MS+6-BA 2.5 mg/L+NAA 0.1 mg/L. The optimum medium

for embryogenic callus induction was MS+6-BA 2.5 mg/L+NAA 0.1 mg/L. The light

quality was 20% blue light + 80% red light (B:R=2:8).

Conclusions At present, the problems of low germination rate, uneven emergence,

unstable flowering period and so on are faced by the sowing and propagation of

Lagerstroemia indica. Through this study, we found that there are two ways of flower

bud regeneration of Lagerstroemia indica. One is that the explants directly form buds

without callus differentiation, and the other is that the explants are induced to form

callus, and finally form buds to complete plant regeneration.When the concentration

of 6-BA, 2,4-D is certain, the induction rate increases first and then decreases with

the increase of NAA concentration, which indicates that auxin in the appropriate

concentration range is beneficial to plant growth.

Acknowledgements This research is supported by the key R & D project of

Ningxia Hui Autonomous Region (special project of modern agricultural science and

technology innovation demonstration area) "new excellent and special flowers

introduction and selection and supporting cultivation technology integration

demonstration".

24

Nutrient Use Efficiency in Crops: A Comprehensive Approach

Virupax C. Baligar1, Zhenli He2, Yuncong C. Li3

1 USDA-ARS, Beltsville Agricultural Research Center, Beltsville MD, U.S.A 2 Indian River Research Education Center -IFAS-Univ of Florida Fort Pierce, FL, U.S.A. 3 Tropical Research Education Center-IFAS -Univ of Florida Homestead, FL, U.S.A.

Abstract This presentation is an over view of significance of improving nutrient use

efficiency in crop to enhance food production for meeting demands of ever-growing

world population and global constraints that could affect achieving such an advance.

In the 21st century, nutrient efficient plants will play a major role in increasing crop

yields. Higher demands for food and fiber by increasing world populations expected

to approach 10 billons by year 2050 further enhances the importance of nutrient

efficient cultivars that are also higher producers in a reduced input system. However,

for achiving needed NUE in crop plants in the future is greatly affected by global

climatic changes (precipitation patterns, drought intensities, light quality/quantity,

greenhouse gases), decline in soil health (deficiency of essential nutrients and

increased toxic elements) and increased occurrences of abiotic (drought, high T and

CO2) and biotic stresses (insect and diseases. Inter- and intra-specific variation for

plant growth and mineral nutrient use efficiency (NUE) are known to be under

genetic and physiological control and are modified by plant interactions with

environmental variables such as soil moisture and deficiency of essential and toxicity

of non-essential elements, temperature, light quality and duration and levels of

atmospheric CO2. Identification of traits such as nutrient absorption transport,

utilization. and mobilization in plant cultivars and their interactions with

environmental variables should greatly enhance nutrient use efficiency in crop plants.

The NUE in the literature is defined in several ways., nutrient efficient plant is

defined; a plant that absorbs, translocate, or utilizes more of a specific nutrient than

another plant under conditions of relatively low nutrient availability in the soil or

growth media. During the last three decades, very limited research has been

conducted to identify and/or breed nutrient efficient plant species or

genotypes/cultivars within species. Complexity of genes involved in NUE for micro

and macronutrients under abiotic and biotic stresses are not well understood. Further,

success in releasing nutrient efficient cultivars has been limited, mainly because of

limited understanding of interactions between plant genetics, environmental variables

25

and adapted management practices. Increased collaborative efforts are needed

between breeders/molecular scientists, physiologists, soil scientists, and agronomists

to evaluate plant nutrient efficiency issues on holistic bases in order to develop

nutrient efficient plant cultivars that could adapt to various types of ecosystems in a

changing climatic conditions and management systems. Hence, during the 21st

century, agricultural scientists have tremendous challenges, as well as opportunities,

to develop nutrient efficient crop plants that can increase plant efficiency for uptake

of applied nutrients and produce higher yields simultaneously adapt to changing

climate and soil fertility and further reduces degradation of soil, water and air quality

and subsequently costs of production.

26

Proliferation and Flowering in vitro of Tissue Culture Seedlings of

Lewisia cotyledon based on Orthogonal Design

Cunyi Ma1; Li Zhang1

College of Agriculture, Ningxia University, Yinchuan, China

Background Lewisia cotyledon, belongs to portulaceae, native to the mountains of

the central west coast of the United States. In recent years, Lewisia cotyledon began

to enter the Chinese market, because of its long flowering period and bright color, it

is popular with the public. However, in the actual production, the seed germination

rate is low and the growth cycle is long, which seriously affects the potted flower

production. Tissue culture and rapid propagation technology has been widely used in

flower production, and the effect is remarkable. The application of tissue culture and

rapid propagation technology in the flower production can promote the rapid

development of its industry.

In bottle flowering refers to the use of in vitro culture method, so that the

flowering process of plants is completed in the container. Flowering in the bottle is

not restricted by climate conditions, which is convenient for cultivation and can

greatly reduce the management cost. However, there are only a few reports on the in

vitro Rapid Propagation of Lewisia cotyledon, and there is no report on the in vase

flowering of Lewisia cotyledon. Therefore, in order to improve the success rate of

Lewisia cotyledon flower cultivation, the rapid proliferation of aseptic seedlings and

in bottle flowering were studied on the basis of in vitro culture of Lewisia cotyledon.

Methods The same size and good growth condition of the aseptic seedlings of

Lewisia cotyledon were selected as experimental materials. Using the orthogonal

design of three factors and three levels L9(33) method, and different concentrations of

sucrose, 6-BA and NAA were added to MS basic medium in order to screen the

optimal proliferation conditions for the aseptic seedling of Lewisia cotyledon.

Different concentrations of 6-BA, NAA and GA3 were added to MS basic medium to

select the optimal conditions for flowering in vitro.

Results The L9(33) orthogonal experiment was designed to select the best conditions

for the proliferation of Lewisia cotyledon. Among the three levels of three different

plant growth regulators, the ninth treatment (A3B3C2) had the best effect. The leaves

27

of Lewisia cotyledon seedlings were thick, the plants were strong, and the average

proliferation multiple reached 3.17 times. L9(33) orthogonal experiment was designed

to select the optimum conditions for flowering in bottles. Among the three levels of

hormone combinations with three different concentrations, the fourth treatment

(D2E1F2) had the most flowering number and the average flowering rate could reach

70%. After inoculation for about 20 days, the plants grew well and began to grow

buds. After 30 days, the plants blossomed.

Conclusions The results showed that MS+0.5mg/LNAA+1.0mg/L6-BA(sucrose

concentration 30g/L) was the best proliferation condition, the highest proliferation

multiple was 3.17 times, the most suitable Flowering in vitro condition was

MS+1.0mg/L 6-BA + 3.0mg/LGA3 + 0.5mg/L NAA, and the average flowering rate

could reach 70%. This research provides theoretical basis and technical guidance for

the efficient propagation and the development of test tube flower in Lewisia

cotyledon.

28

Plant growth regulators and EDTA improve phytoremediation

potential and antioxidant response of Chenopodium

ambrosioides L. in a Cd-spiked soil

Amin Ullah Jan1, Allah Ditta2,3

1 Department of Biotechnology, Faculty of Science, Shaheed Benazir Bhutto University

Sheringal Dir Upper, 18000, Khyber Pakhtunkhwa, Pakistan 2 Department of Environmental Sciences, Shaheed Benazir Bhutto University Sheringal,

Dir Upper, 18000, Khyber Pakhtunkhwa, Pakistan 3 School of Biological Sciences, The University of Western Australia, 35 Stirling

Highway, Perth, WA 6009, Australia

Objectives Soil pollution due to potentially toxic elements is a worldwide challenge

for health and food security. Chelate assisted phytoextraction along with the

application of plant growth regulators (PGRs) could increase the phytoremediation

efficiency of metal contaminated soils. The present study was conducted to

investigate the effect of different PGRs (GA3 and IAA) and synthetic chelator

(EDTA) on growth parameters and Cd phytoextraction potential of Chenopodium

ambrosioides grown under Cd-spiked soil.

Methods GA3 (10-7 M) and IAA (10-5 M) were applied four times with an interval of

ten days through foliar spray, while EDTA (40 mg kg-1 soil) was once added to the

soil.

Results The results showed that Cd stress significantly decreased fresh biomass, dry

biomass, total water contents, and photosynthetic pigments as compared to control.

Application PGRs significantly enhanced plant growth and Cd phytoextraction.

Combined application of GA3 and IAA with EDTA significantly increased Cd

accumulation (6.72 mg kg-1 DW) and bioconcentration factor (15.21) as compared to

C1 (Cd only). The same treatment significantly increased chlorophyll, proline,

phenolic contents, and antioxidant activities (CAT, SOD, and POD) while MDA

contents were reduced. In roots, Cd accumulation showed a statistically significant

and positive correlation with proline, phenolics, fresh biomass, and dry biomass.

Similarly, Cd accumulation showed a positive correlation with antioxidant enzyme

activities in leaves. Chenopodium ambrosioides showed Cd hyperaccumulator

potential, based on bioconcentration factor (BCF) > 1.

29

Conclusions In conclusion, exogenous application of GA3 and IAA reduces Cd stress

while EDTA application enhances Cd phytoextraction and ultimately

phytoremediation potential of Chenopodium ambrosioides.

Acknowledgements This research did not receive any specific grant from funding

agencies in the public, commercial, or not-for-profit sectors.

30

Plants buffer against high light stress-induced transcript of

CsGA2ox8 via alternative splicing to fine-tune gibberellins

level and maintain hypocotyl elongation

Bin Liu1,2, Shuo Zhao1, Pengli Li1, Yilu Yin1, Qingliang Niu1,

Jinqiang Yan3, 4, Danfeng Huang1

1 School of Agriculture and Biology, Shanghai Jiao Tong University, Key Laboratory of Urban

Agriculture (South), Ministry of Agriculture, Dongchuan Road, Shanghai 200240, China 2 Department of Plant Genomics, Centre for Research in Agricultural Genomics

(CRAG), CSIC-IRTA-UAB-UB, Bellaterra 08193, Spain 3 Vegetable Research Institute, Guangdong Academy of Agricultural Sciences,

Guangzhou 510640, China 4 Guangdong Key Laboratory for New Technology Research of Vegetables, Guangzhou

510640, China.

Background In plants, alternative splicing (AS) is markedly induced in response to

environmental stresses, but it is unclear why plants would generate multiple

transcripts under stress conditions.

Methods In this study, RNA-Seq was performed to identify AS events in cucumber

seedlings grown under different light intensities. We characterized the expression of

target genes by quantitative real-time PCR. Functional analysis was done with

transgenic plants. GA contents were measured by Liquid chromatography-mass

spectrometry.

Results We identified a novel transcript of the gibberellin (GA) deactivation enzyme,

Gibberellin 2-beta-dioxygenase 8 (CsGA2ox8). Compared with canonical

CsGA2ox8.1, the isoform CsGA2ox8.2 had an intron retention between the second

and third exons. Functional analysis proved that the transcript of CsGA2ox8.1, but

not CsGA2ox8.2, played a role in the deactivation of bioactive GAs. Expression

analysis demonstrated that both transcripts were upregulated by increased light

intensity, but the expression level of CsGA2ox8.1 increased slowly when the light

intensity was greater than 400 µmol·m-2·s-1 ppfd (photosynthetic photon flux density),

while the CsGA2ox8.2 transcript increased rapidly when the light intensity was

stronger than 200 µmol·m-2·s-1 ppfd.

Conclusions Our findings provide evidence that plants might fine-tune GA levels by

buffering against the normal transcript of CsGA2ox8 through AS.

31

Acknowledgements This study was supported by The National Key Research and

Development Program of China (2019YFD1000300), the International Postdoctoral

Exchange Fellowship program from China Postdoctoral Council (20170053); the

Technology System Construction of Modern Agricultural Industry of Shanghai

(19Z113040008), and the Presidential Foundation of Guangdong Academy of

Agricultural Sciences (BZ201901).

32

Seeds priming technology as a key strategy to increase crops

production under adverse environmental conditions

Daniel Garcia1, Shuo Zhao1, Samiah Arif1, Yinglei Zhao2,

Lin Chau Ming3, Danfeng Huang1,

1 School of Agriculture and Biology, Shanghai Jiao Tong University (SJTU), Shanghai

-200240, China 2 College of Bio Systems Engineering and Food Science, Zhejiang University, Hangzhou

310058, China 3 Horticulture Department, Faculty of Agronomic Sciences (FCA), São Paulo State

University Julio de Mesquita Filho (UNESP), 18610-307, Botucatu, São Paulo, Brazil

Corresponding author:Tel.:+8618621759810. E-mail address: [email protected]

Objectives Faced with environmental adversities, and the increasing food demand

around the globe, more attention has turned to improve crop production by efficient

strategies. Thus, we aimed to study hydropriming, osmopriming, chemo/biopriming,

thermopriming, hormopriming, nanopriming, and hybrid priming methods on the

germination and growth performance of several crops under environment adversities.

Methods Physiological, biochemical, and molecular changes on seeds subjected to

priming methods and allowed to germinate under environment adversities were

detected in a wide number of reports. The main databases were visited: PubMed,

Elsevier, MDPI, Springer among others.

Results All the seven priming methods demonstrated promising results related to

enhance the crop production traits, alleviation of aging damage, enhance chilling and

heat tolerance, improve phytopathogen tolerance, mitigate the effects of salinity,

heavy metal, and flooding, enhance seeds storage prolongation and increase drought

tolerance. The findings shed light on the use of priming technology as a key strategy

to increase crop production via stress-tolerance faced to the predicted environment

adversities and the ever-increasing food demand.

Conclusions The molecular point of view must be further investigated on seeds

subjected to priming methods, in order to understand the specific influence of each

biological mechanism on the production of crops under stress conditions. Moreover,

we strongly recommend the combined priming methods, called here hybrid priming,

generating thus, new approaches about the complex biological mechanism changes in

the enhancement of crop productivity under environment adversity.

33

Effects of greenhouse new/waste rockwool and nutrient

on growth and quality of pakchoi

Xiaotao Ding, Shuailei Zhang, Yingqi Chu, Lizhong He,

Hong Wang, Qiang Zhou, Jizhu Yu

Shanghai Key Lab of Protected Horticultural Technology, Shanghai Academy of

Agricultural Sciences, Shanghai 201106, China

Objectives The effective utilization of waste rock wool and drain nutrient is an

important problem that perplexes the development of modern greenhouse. The

objective of the experiment was to find method to effective use waste rockwool and

drain nutrients.

Methods the experiment was designed with four treatments: 1. new rockwool with

new nutrient; 2. new rockwool with waste nutrient; 3. waste rockwool with new

nutrient; 4. waste rockwool with waste nutrient. All seedlings were watered with

nutrient of EC1.8 dS/m and pH of 5.8. The four treatments in this study were

arranged in a randomized complete block design. The experiment was replicated

three times with one experimental unit (twenty plants) per treatment. In the

experiment, the growth changes of pakchoi in the cultivation of new and waste

rockwool, and new and waste nutrient solution, as well as chlorophyll content,

photosynthesis, yield and important quality parameters were measured.

Results The results showed that the growth and photosynthesis of pakchoi treated

with waste rockwool were clearly better than that treated with new rockwool, yield of

waste rockwool with new nutrient and waste rockwool with waste nutrient treatments

were significantly higher than new rockwool with new nutrient and new rockwool

with waste nutrient treatments of 13.5% and 20.2%,of 14.4% and 21.2%, which may

be related to the more nutrients in waste rockwool. There was no clear difference in

quality, which the content of soluble sugar, total phenol and flavonoids were not

significant difference for different treatments.

Conclusions The treatments of waste rockwool and waste nutrient solution had not

significantly reduced quality of pakchoi, and the content of nitrate and nitrite did not

show drastic changes. This experiment provides an effective method for the recycling

of waste rockwool and drain nutrients in modern greenhouse.

Acknowledgements This work was supported by Shanghai Agriculture Applied

Technology Development Program, China (Grant No. T20180405).

34

Purification ability from constructed wetlands treating sewage

treatment plants effluent and microbial community

succession in epiphytic biofilms

Zheng Qiu, Songhe Zhang*, Yan Ding, Wenjun Zhang, Lixue Gong,

Qiang Yuan, Xiaoying Mu, Dongwang Fu

Ministry of Education Key Laboratory of Integrated Regulation and Resource Development on

Shallow Lakes, College of Environment, Hohai University, Nanjing, 210098, China.

*Corresponding author: Songhe Zhang Email: [email protected]

Abstract Sewage treatment plants effluent is a large source of pollution and it’s

important to treat it in a clean way. In aquatic environments, submerged macrophytes

play important roles in bioremediation of wastewater and microbes in epiphytic

biofilms play crucial roles in the pollutants removal in constructed wetlands.

However, the impacts of treated sewage effluents on nutrients removal and epiphytic

microbial community structures in wetlands dominated by submersed macrophytes

remain to be investigated. In this study, concentrations of nutrients in three kinds of

simulated effluents were used according to the first-level standard A and B and

second-level standard in discharge standard of pollutants for municipal wastewater

treatment plant in China (GB18918-2002). M. Spicatum with the same growth stage

and artificial plants (made by polyvinyl chloride) with similar morphological

characterizations (needle leaves) were used in this experiment. While under

second-level standard, the leaf surface of artificial plants and M. Spicatum was

almost completely covered by microorganisms. Compared to M. Spicatum, artificial

plants had higher NH4+-N, TN, COD and TP removal rates in wetlands receiving

treated sewage effluents. Taken together, artificial plants systems had better

performance in nutrients removal than M. Spicatum systems in general. The

α-diversity values of bacterial and eukaryotic community in attached biofilms were

significantly higher in MS systems than in AP systems, revealing microbial

population richness was higher in epiphytic biofilms on M. Spicatum than artificial

plants. However, there were no significant difference in the alpha diversity values

caused by different water qualities or time variable. Both turnover (species

replacement) and nestedness (species loss) contributed to the construction of

microbial community structure, whereas beta-diversity patterns in constructed

wetlands mainly resulted from species replacement rather than species loss. Organic


35

matter decomposition-related microbes were stimulated in MS systems bacteria in M.

Spicatum systems. Phyla Ciliophora occurred in a higher frequency in M. Spicatum

systems than artificial plants systems, which can be ascribed to lower nutrients

removal rates in M. Spicatum systems. Network analysis revealed that there were

more complex interactions among environmental parameters, bacteria and eukaryotes

in M. Spicatum systems than in artificial plants. Those results demonstrate that AP

can provide surface for biofilm growth and have roles similar to submersed

macrophytes in nutrients removal while treated sewage effluents can cause damage to

submersed macrophytes, highlighting artificial plants can be used in effluent deep

treatments and the strict discharge standards should be taken to reduce the nutrients

in treated sewage effluents, for aims to protect the ecological systems.

Keywords Treated sewage effluents; Beta-diversity; Microbes; Shannon index;

Co-occurrence network

36

Impacts of water flow and suspended sediment on microbial

community in epiphytic biofilm on two

submerged macrophytes

Hezhou Chen, Bing Han, Songhe Zhang*

Ministry of Education Key Laboratory of Integrated Regulation and Resource Development on

Shallow Lakes, College of Environment, Hohai University, Nanjing, 210098, China. *Corresponding author: Dr. Songhe Zhang E-mail: [email protected]

Abstract Understanding the interaction of water flow, suspended sediment (SS) and

submerged macrophytes is of great significance to water ecological restoration and

management. In this lab-scale water tank experiment, the influence mechanism of

different submerged macrophytes and water flow on the removal of SS was studied

with a focus on understanding the effects of water flow and SS on the structure and

microbial composition in the epiphytic biofilm of M. verticillatum and V. natans. It

was found that the decreasing trend of water turbidity can be elucidated by the

logarithmic model (R2>0.93, P<0.01) within one day regardless of water flow and

plant types. The turbidity reduction rate was higher in treatments with plants than

without plants in static water (96.2%>91.6%), but an opposite trend was observed

under flowing water (95%<98.2%). The size of particles was mainly below 500 μm

(72.7%-94.6%) on leaves of two submerged macrophytes, and among four treatments,

particles was the smallest but its amount was the biggest on M. verticillatum leaf

under water flow. Confocal laser scanning microscopy images revealed that the mean

values biofilms thickness ranged from 10.18 to 28.36 μm. Mantel correlation

analyses revealed that water flow was significantly correlated with the relative

abundance of top ten bacterial (r=0.6433, p=0.0284) and eukaryotic (r= 0.6343,

P=0.0285) order taxa in epiphytic biofilm of two submerged macrophytes; SS

decreased such correlation. Principal co-ordinates analysis of bacterial and eukaryotic

showed that the bacterial communities in epiphytic biofilm of M. verticillatum and V.

natans. were significantly different from those in spiked SS in treatment without

water flow, but the eukaryotes in spiked SS and epiphytic biofilm had similar

community composition in treatments with water flow, suggesting that the eukaryotes

on sediment may be transferred to the leaves due to water flow. SS increased

epiphytic biofilm bacterial diversity, but inhibited eukaryotic diversity, except

inhibited bacterial diversity and promoted eukaryotic diversity in V. natans under


37

static water. Results of co-occurrence networks shows that SS increased but water

flow decreased the number of keystone microbes. Our results highlighted that water

flow and SS have different mechanism on microbial community in epiphyte biofilm

on submersed macrophytes.

Keywords Suspended sediment; Water flow; Submerged macrophytes; Epiphytic

biofilm; Microbial community

38

Aeration enhanced nutrient removal and increased microbial

diversity during Hydrilla verticillata decomposition

in wetlands in winter

Xin Lv, Tiantian Zhou, Songhe Zhang, Lisha Zhang,

Xiaoying Mua, Ziqiu Zhang, Qiang Yuan

a Ministry of Education Key Laboratory of Integrated Regulation and Resource

Development on Shallow Lakes, College of Environment, Hohai University, Nanjing

210098, China.

Corresponding author: [email protected] (S.H. Zhang)

Abstract Aquatic environments usually support high macrophyte productivity, while

after senescence, abundant nutrients can be rapidly released, causing the potential

deterioration of the water environment. The decay of aquatic macrophyte causing low

performance in nutrients removal is remain unsolved in wetland. Mechanical aeration

has been reported to reduce the total concentration of nitrate and phosphate in

wastewater. In present study, nutrient release, extracellular activities and microbial

communities were investigated during H. verticillata decomposition in a natural (NA)

or aerated (AA) environment for 206 days. A small aeration pump was placed 5 cm

below the water surface to pump air at 1 L of air/min in the AA groups. The results

showed aeration increased the dissolved oxygen concentration and decreased the

nutrient content in the overlying water, enhanced extracellular activities in biofilms

and decreased the C, N and P content in plant residues in comparison to what was

observed in the NA treatment. Humic acid-like and tryptophan-like substances

significantly increased during H. verticillata decomposition, which were originated

from organic matter containing N and P in the overlying water, especially in the AA

treatment. The bacterial 16S rRNA (V3-V4 region) and eukaryotic 18S rRNA (V4

region) genes in surficial sediments and biofilms were analysed at nine sampling

times. The microbial communities in biofilms generally differed from those in

surficial sediments. The bacterial communities in biofilms shifted and became similar

to those in surficial sediments over time. Aeration promoted relative abundance of

aerobic bacteria but suppressed facultatively and obligate anaerobic bacteria. The

dominant phylum in samples were Stramenopila, Alveolata, Rhizaria, Metazoa, Fungi,

Viridiplantae and Apusozoa. Aeration stimulated relative abundance of Metazoa but

suppressed Viridiplantae in sediments. Network analysis showed complex microbial

interactions in the leaching stage and aeration altered the microbial interactions

39

during decomposition. The eukaryotic community was more stable than the bacterial

community, especially among bacteria in sediments, while aeration obviously

disturbed the bacterial community during decomposition. Our results highlight that

aeration can enhance nutrient removal by stimulating microbial activity and shaping

the microbial community during H. verticillata decomposition.

Keywords Humic acid; nutrients; eukaryotic community; decomppsition

40

Detection of chlorophyll content in tomato leaves based

on portable hyperspectral imaging technique

Minghua DU, Lu Meng, Longguo Wu*

School of Agriculture Ningxia University, Yinchuan Ningxia 750021

Background Nitrogen is the primary factor affecting tomato growth and yield. In

production, excessive nitrogen application and excess nitrogen application can inhibit

the healthy growth of tomatoes. Therefore, it is imperative to adopt scientific

nitrogen nutrition diagnosis technology, establishing a set of reasonable diagnostic

indicators and recommend fertilization technology system. In recent years,

hyperspectral imaging technique has been regarded as one of the most promising

diagnostic methods. As a nondestructive analysis technique, it can be applied to plant

nutrition detection more quickly and effectively. Many scholars at home and abroad

have realized the nondestructive testing of nitrogen, phosphorus, potassium,

chlorophyll and other nutrient elements of crops based on hyperspectral imaging

technology. At present, there are few studies on nitrogen content detection in tomato

leaves by using hyperspectral imaging technology. Therefore, on the basis of

previous studies, portable hyperspectral imaging technology was used to study and

detect nitrogen content in tomato leaves.

Methods The average spectral reflectance data (400-1000nm) of 82 tomato leaf

samples were extracted and pretreated with 7 kinds of original spectral data (mean

smoothing, gaussian filtering, median filtering, convolution smoothing, normalization,

baseline calibration, and standard normalization(SNV). Feature spectral information

was extracted by weight coefficient method(BW), variable elimination

transformation without information method (UVE), competitive adaptive repeated

weighting method (CARS) and continuous projection algorithm (SPA). Linear

regression (MLR, PCR, PLSR) and nonlinear regression (SVR) were used to

construct the quantitative prediction model of chlorophyll in tomato leaves.

Results In the pretreatment comparison, the prediction ability of SNV pretreatment

model was higher than others.Five kinds of normalized pretreatments were compared

and analyzed, and the PLSR model established by normalized -3(average

normalization) had the best effect. The PLSR model of the original spectrum and

other pretreated spectra were compared and analyzed, and the PLSR model

41

established by SNV was selected. Compared with other feature bands selected,

CARS was the best method for selecting feature wavelengths. Eight characteristic

wavelengths (732, 796, 946, 953, 957, 968, 983 and 994nm) were extracted by CARS

method and applied to establish a quantitative prediction model of chlorophyll in

tomato leaves. The model of MLR, PCR, PLSR and SVR were used to compare the

models of characteristic wavelengths extracted by CARS, and it was found that the

model established by MLR, PCR, PLSR was superior to the model established by

SVR. Among the models established by linear regression, the effect of MLR model is

better than PCR and PLSR. PCR and PLSR models established by linear regression

have basically the same correlation coefficient and root mean square error.

Conclusion Our study shows that the noise interference in spectral information can

be improved to a certain extent by the selection of pretreatment methods. Eight

characteristic wavelengths (732, 796, 946, 953, 957, 968, 983, 994nm) extracted by

CARS for tomato leaf chlorophyll quantitative model is set up, has good prediction

effect. The linear regression model of chlorophyll in tomato leaves obtained a higher

prediction effect. The correlation coefficient and RMS error of MLR of the optimal

prediction model were 0.794 and 3.492, respectively. On this basis, the visual

distribution was carried out, and finally MLR model was selected to predict the

chlorophyll in tomato leaves online, which also provided reference for the rapid

detection of other indicators of tomato plants.

Keywords Hyperspectral imaging; Tomato leaves; Chlorophyll; Nondestructive

testing

42

Research of Volatile Components on Champagne and

Japanese Peppermint Stem and Leaf Tissues

Liu Xuanxuan, Zhang Haoyu, Zhang Guangdi

School of Agriculture Ningxia University, Yinchuan Ningxia 750021

Background Peppermint belongs to the genus Mentha haplocalyx Briq. It is widely

distributed and mainly concentrated in the temperate regions of the northern

hemisphere. Peppermint has the function of clearing heat and reducing fire. It can be

used for wind-heat and cold, sweating and antipyretic, calming and analgesic,

anti-inflammatory and antibacterial, etc. Efficacy, this test aims to accurately locate

the extraction of peppermint essential oil, the use of processing materials such as

flavors and fragrances, condiments, tea beverages, etc. through the specific

characteristic volatile substances in different peppermint stem and leaf tissues, and

provide methods for the identification of peppermint varieties.

Methods In this research, gas chromatography-ion mobility spectrometry (GC-IMS)

technology was used to detect and analyze the volatile components in the stems and

leaves of Champagne and Japanese peppermint. In the two types of peppermint, 39

volatile compounds were identified. It mainly included alcohols, ketones, aldehydes,

furans, esters and terpenes. Among the stem and leaf tissue of Champagne mint, there

are 28 volatile compounds are identified. In Champagne mint leaves, the main

volatile substances were 3 -Octanol eucalyptol, linalool, trans-2-hexen-1-ol,

2-heptanone, etc; In Champagne peppermint stems, the main volatile substances

were 2,3-butanedione, etc.; A total of 38 volatile compounds have been identified in

Japanese mint. The main compounds in the leaves were 3-octanol, 1-octen-3-ol,

3-octanone, ethyl isobutyrate, linalool, and butyraldehyde, Α-pinene, 1-pentanol, etc.

In Japanese peppermint stems, the main volatile substances were ethyl acetate,

2-n-pentyl furan, pentyl acetate, 2-nonanone, isobutyl acetate, etc. Compare between

Champagne peppermint and Japanese peppermint leaves showed that

2,3-butanedione was a unique volatile compound in Champagne peppermint.

Compared the stems of two peppermints, 2-nonanone, 2-n-pentylfuran, and ethyl

hexanoate. Ester, butyl acetate, etc. were unique volatile substances in Japanese

peppermint, but they were not detected in Champagne peppermint.

Results Through PCA and visual heat map cluster analysis, it could be known that

43

the volatile compounds in the stem and leaf tissue of each peppermint were different

and could be obtained. Obviously, this test aimed to accurately locate the extraction

of peppermint essential oil, the use of processing materials, such as flavors and

fragrances, condiments, and tea beverages, and to provide methods for the

identification of peppermint varieties.

Conclusions Through the above analysis, it can be seen that Champagne and Japanese

peppermint, these two types of compounds are the main volatile substances in the

extraction of peppermint essential oil. In addition, there are also some terpene volatile

compounds such as α-pinene, which have certain properties. The anti-inflammatory,

anti-viral, antibacterial and other effects of the peppermint are relatively high in the

peppermint stems and leaves; through the visual heat map and PCA analysis, the

volatile organic compounds in the two peppermint leaves are very different in

composition and content. If passed More samples are modeled, and this result will also

provide a theoretical basis for the identification of mint leaves and stems in organic

matter and the identification of unknown mint samples. Through the above results, the

use of the characteristic volatile compounds in different tissues of mint can provide

scientific and reasonable suggestions and support for the deep processing of mint, such

as food flavors, spices and condiments in the future.

44

Study on Visualization Distribution of Chlorophyll in Tomato

Leaves under brackish irrigation Based on

Hyperspectral Imaging Technology

Lu Meng, Jie Zhang, Tian Yang, LongGuo Wu*

School of Agriculture, Ningxia University,Yinchuan 750021,China

Background Ningxia is located in the arid northwest of China, which riches in

brackish water and saline water resources, using brackish water for irrigation to

alleviate the crisis of freshwater resources. The nitrogen content of crop leaves

reflects the response of brackish water to the organism, and provides guidance for the

rational application of brackish water in the future. Hyperspectral imaging

technology has been widely used in the estimation of crop physiological and

biochemical indicators,which is faster and more non-destructive than the chlorophyll

meter.

Methods The test tomatoes were selected from the "Bomei No.2" variety, with a total

of 108 tomato seedlings. The seedlings were transplanted when 3-4 cotyledons were

expanded, and the cultivation substrate pot was used for planting. Each pot was

planted with 4 tomato seedlings. The experiment was designed with 9 treatments in

follows, control group CK: distilled water; A:0.2 g/L NaCl solution; B:0.4 g/L NaCl

solution; C:0.6 g/L NaCl solution; D:0.8 g/L NaCl solution; E:1.0 g/L NaCl solution ;

F:1.2 g/L NaCl solution; G:1.4 g/L NaCl solution; H:1.6 g/L NaCl solution,and each

treatment was repeated 3 times. In the early stage, the optimum irrigation amount was

determined to be 550ml, after planting, irrigation should be done every 2 days. After

30 days of planting, the canopy leaves of tomato were picked. A total of 150 pieces

were collected in the experiment, and the portable chlorophyll tester and portable

hyperspectral imaging equipment were used for chlorophyll measurement and image

collection immediately.

Results The results show that the outlier elimination optimizes the model and

optimizes the original spectrum for data analysis. PLSR method was used to model

the characteristic wavelength extracted by different methods and to optimize the

characteristic wavelength extracted by SPA. On this basis, the models of MLR, PCR,

PLSR, SVR, ANN were used to model the characteristic wavelengths extracted by

SPA, and the different modeling effects are compared and analyzed. The models of

45

MLR, PCR, PLSR are preferably selected. The optimal characteristic wavelengths

were 392, 465, 686 and 760nm, the prediction correlation coefficient of the optimal

model was 0.896, and the prediction root-mean-square error RMSEP was 1.111.

Finally, the PLSR model was optimized to predict the chlorophyll content of tomato

by visualizing three linear models.

Conclusions 1.Using Euclidean distance and Mahalanobis distance combined with

Monte Carlo method to eliminate abnormal samples, a robust model can be

established. 2.The tomato leaf chlorophyll quantitative model established by the 4

characteristic wavelengths (392, 465, 686, 760nm) extracted by the SPA , the method

has a good predictive effect. 3.The linear regression model of tomato leaf chlorophyll

has better prediction effect than the nonlinear regression model. The correlation

coefficient and root mean square error of the optimal prediction model are 0.896 and

1.111 respectively; on this basis, visualize the distribution, and finally choose the

PLSR model for online prediction of tomato leaf chlorophyll.

46

Comparison and Screening of Total RNA Extraction

Methods from Fennel Leaves

Wang Lin1, Wang Xiaomin1,2,3,4*, Li Xia1, Guo Liwen1,

KongWeikang1, PanBingqing1

1 School of Agriculture, Ningxia University 2 Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia 3 Ningxia Facility Horticulture (Ningxia University) Technology Innovation Center 4 Ningxia Modern Facility Horticulture Engineering Technology Research Center

Background Fennel is a kind of 1-year-old and 2-year-old herb of Umbelliferae,

which originated in Mediterranean region. It has been planted all over the world

because of its strong adaptability. It has been cultivated for more than 1000 years in

China and distributed all over the country. In recent years, research on fennel mainly

focused on its physiological active components, inducing plant seed germination,

extracting volatile oil components and so on, but the molecular regulation mechanism

of fennel was still unclear. It is necessary to extract high-quality RNA in order to

explore the related genes and elucidate their expression and regulation. Extracted

high-quality RNA from plant tissues was the premise of molecular biology research,

such as gene cloning, northern analysis, RT-PCR and cDNA library construction,

because of differences in plant tissue composition and environmental stress, different

metabolites are produced during plant growth, such as proteins, polysaccharides and

polyphenols etc. It was easy to coprecipitated with RNA, and interfered with the

extraction and purification of RNA, then the quality of total RNA was affected. So

the methods of total RNA extraction of plants should be screened and compared.

Methods Hexadecyl trimethyl ammonium bromide(CTAB) method, modified CTAB

method, CTAB water saturated phenol method, Tris-boric acid method,

cyanoguanidine isosulfate method, modified cyanoguanidine isosulfate method,

Trizol method, and modified Trizol method were used to extracted RNA from leaves

of fennel seedlings at seven leaves old. The concentration, purity and integrity of

total RNA from fennel leaves were determinated by nucleic acid and protein detector

as well as agarose gel electrophoresis.

Results The quality of RNA extracted by modified CTAB method was the best, and

the concentration was 1481.9 μg/μL, the OD260/280 was 1.82, OD260/230 was 2.12, the

yield was higher, and the strips were clear and intact without tailing. The quality of

47

RNA extracted by Trizol method, modified Trizol method and cyanoguanidine

isosulfate method was higher, but the integrity was poor, and the former two methods

had higher cost, while the latter was lower. Both of the purity and integrity of RNA

extracted by Tris-boric acid method were the lowest. The effects of total RNA

extraction by CTAB method, CTAB water saturated phenol method and modified

cyanoguanidine isosulfate method were poor.

Conclusions Our study indicated that CTAB was selected as the most suitable

method for extracting RNA from fennel leaves, which laid a foundation for

subsequent molecular biology research and provided some reference for RNA

extraction from similar plants.

48

Analysis of Flavor Compounds in Grapefruit Mint

Leaf Stem Based on GC-IMS

Yufen Xie1,2, Haoyu Zhang1,2, Wenli Yang1,2, Kunming Zhang2,4, Yuelin Ma2,4,

Xiaoyun Liu2,4, Xiaoyan Yu3*,and Guangdi Zhang1,2,3*

1 School of Agriculture Ningxia University,Yinchuan 750021, China 2 Ningxia Key Laboratory for FoodMicrobial-applications Technology and Safety

Control,Yinchuan 750021,China 3 Technological Innovation Center of Protected Horticulture (Ningxia University) in

Ningxia, Yinchuan 750021, China 4 School of Food and Wine Ningxia University,Yinchuan 750021, China

Background Grapefruit mint, (Mentha Suaveolens × Piperita) is a perennial herb of

the genus Mentha in the labiform family. It is one of the new varieties of mint and has

a very strong grapefruit taste. As a common Chinese traditional medicine, peppermint

USES whole grass as medicine. It has the functions of dispelling wind and heat,

clearing the head, benefiting the throat, penetrating the rash, soothing the liver and

activating the qi. Volatile essential oil extracted from the stems and leaves of mint has

the effects of fragrance, refreshment, brain awakening, solution table and

antibacterial, etc. It has been widely used in the fields of daily chemicals, food and

drugs. Modern medicine has also proved that peppermint has anti-inflammatory and

analgesic effects, anti-fungus, anti-tumor, anti-oxidation and anti-radiation effects.

Grapefruit mint, as a medicinal and edible plant, has a wide range of pharmacological

effects, but the difference between the volatile components of the stem and the leaf is

not clear.

Methods Grapefruit mint samples were collected from cuttings at the age of 4-5

months and planted on floating plates in the closed loop symbiosis system of

fish-flower (grapefruit mint). The leaves and stems of grapefruit mint samples were

treated separately immediately after harvesting for GC-IMS analysis. In this paper,

the differences of volatile components in grapefruit mint leaves and stems from

fish-flower symbiotic closed-loop system were analyzed by gas chromatography-ion

migration spectrum (GC-IMS).

Results We found a total of 36 qualitative volatile substances, including alcohols,

aldehydes, ketones, esters, terpenes, ethers and heterocycles, were identified in the

leaves and stem tissues of grapefruit mint. There were 12 alcohols, 6 aldehydes, 6

49

esters, 6 ketones, 2 ethers, 1 terpene and 3 heterocycles. In alcohols, aldehydes, esters,

ketones, ethers, heterocyclic substances, trans-2-hexenol, geraniol and linalool,

n-hexanal, methyl 2-methylbutyrate, iso-amyl acetate, acetone, 1-octene-3-ketone,

dimethylsulfur and 2-acetylfuran peak volume is large. PCA analysis showed that the

composition of volatile matter in grapefruit mint leaves and stems was significantly

different. The volume of linalool, dimethyl trithiol and ethyl acetate was larger in

leaves and stems, while the volume of trans-2-hexenol, geraniol and eucalyptus oil

was larger in leaves. The peak volume of iso-valeral and butyraldehyde in stem was

larger.

Conclusions GC-IMS was used to analyze the volatile compounds in the leaves and

stems of grapefruit mint. After removing the indeterminate and dimer, there were 36

kinds of qualitative compounds, including alcohols, aldehydes, ketones, esters,

terpenes, ethers and heterocycles. Among them, the volume of trans-2-hexenol,

geraniol, linalool, n-hexyl aldehyde, methyl 2-methylbutyrate, iso-amyl acetate,

acetone, 1-octene-3-ketone, dimethylsulfur and 2-acetylfuran peaks in grapefruit lotus

leaves and stems was relatively large, while that of trans-2-hexenol, geraniol and

eucalyptus oil were relatively high. The iso-amyl aldehyde and butyraldehyde were

higher in the stem. As the main volatile substance, linalool has antiviral and

bacteriostatic effects. Eucalyptus, acetone, dimethyl-sulfur, pinene, 2-ethyl furan all

have inhibitory effects on bacteria. Geraniol has insect repellent and antibacterial

properties. It is clinically used in the treatment of hepatitis and colitis, and has the

effects of alleviating neuroinflammation, inhibiting tumor growth and lowering blood

sugar. Through this study, the differences of volatile substances in the stem and leaf

tissues of grapefruit mint were preliminarily verified, which provided a theoretical

basis for the better application of the stem and leaf tissues of grapefruit mint in

medicinal materials, and also laid a certain foundation for its further application

research.

50

Comparative Analysis of Volatile Components in Leave

and Stem of the Mentha spicala by GC-IMS

Yang Wenli, Zhang Haoyu, Zhang Guangdi

Background The fish-vegetable symbiosis cycle system has realized the ecological

effects of water saving, emission reduction and resource reuse, and is considered to

be a modern agricultural production model with great innovation and sustainable

development potential [10]. Applying spearmint to the fish-flower closed-loop

symbiosis cycle system, using hydroponics technology, it can quickly and efficiently

utilize the metabolites and various mineral nutrients of fish with water as a carrier,

and at the same time achieve zero emissions to the environment. It is a modern

agricultural model that combines high-efficiency aquaculture and hydroponic

planting, which can fully reflect the maximum utilization of resources and the

optimization of benefits. Gas chromatography-Ion mobility spectrometry (GC-IMS)

is a detection technology that combines ion mobility spectrometry and gas

chromatography. It has the advantages of fast detection speed, convenient operation,

and portable equipment. A convenient tool for chemical analysis of food flavor. At

present, it has been widely used in food flavor analysis, quality inspection and other

fields. Compared with the traditional GC-MS method, the GC-IMS method does not

require vacuum, the sample does not need to be enriched and concentrated, and the

temperature for analysis is lower, and some have better thermal stability. Poor

volatiles can be detected more realistically, and the volatile organic components in

the tested sample can be quickly detected by direct headspace sampling. It is used to

identify the authenticity, freshness, shelf life, variety and quality of samples.

Methods Gas chromatography ion mobility chromatography (GC-IMS) was used to

analyze difference of the volatile components of the leaf and stem of Mentha Spicala

which planting in the fish - flower (Spearmint) closed loop symbiosis cycle

system.The GC-IMS Library Serach software was used for quantitative

analysis,according to the retention time of volatile substances by gas chromatography

and IMS migration time. There were 30 volatile substances in stem and leaf tissues of

Spearmint ,which were alcohols, Ketones, esters, aldehydes, sulfur, heterocyclics and

terpenes, among which 10 kinds of were alcohols, accounting for 33.33%.

Results Accordingly, the main volatile components in the leaves and stems of

51

Spearmint are alcohols, ketones, and aldehydes, with the content of alcohols being

the highest. And the overall concentration of volatile components in the leaves is

higher, such as 2-methylfuran, hexanal, 1-pentanol, butyraldehyde, ethanol,

3-methylbutanal, dimethyl sulfide, 2-Ethyl furan, 2-heptanone, benzaldehyde, α-pinene,

isobutanol, ethyl isobutyrate, 1-hexanol, trans-2-hexen-1-ol, linalool, and 3-octyl, all of

these, the content of leaves is much higher than that of stems. In addition,

2-acetylfuran, n-hexanal, ethyl isobutyrate, 1-pentanol, butyraldehyde, ethanol,

isovaleraldehyde, etc. are unique to leaves. And, only a few substances are more

abundant or unique in the stem.

Conclusions Wang Zhonghua and other studies have shown that the spearmint stem

and leaf powder contains a variety of organic acids and flavonoids, which are

beneficial to improve the body’s immune function. After the oil is extracted, the

spearmint still has residual alcohols, ketones and esters. These components can

promote the secretion of intestinal glands, promote the digestion of nutrients,

promote the appetite of animals, increase feed intake, optimize the utilization and

metabolism of nutrients, and the fish-flower (mint) closed-loop symbiosis cycle The

volatile components of the leaves and stems of spearmint in the hydroponic system

are mainly alcohols, ketones, aldehydes and esters. In order to improve the utilization

value of the leaves and stems of spearmint, the spearmint stem and leaf powder after

oil extraction can be applied to livestock and poultry production to increase the added

value of spearmint planting.

52

The Identity of Pathongen Causing Tomato

Powdery Mildew in Yinchuan

Zhou Pengze1, Wang Xiaomin1, 2, 3, 4, Kong Weikang1, Wang Lin1,

Bai Shengyi1, Gao Yanmin1, 2, 3, 4, Li Jianshe1, 2, 3, 4

1 School of Agriculture, Ningxia University. 2 Ningxia Modern Facility Horticulture Engineering Technology Research Center. 3 Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia. 4 Ningxia Facility Horticulture（Ningxia University）Technology Innovation Center.

Background Tomato powdery mildew is a global fungal disease. After Salmon

discovered in 1900 that the pathogen causing tomato powdery mildew in Italy was

Erysiphe polygoni DC. Then tomato powdery mildew in Japan, the Netherlands, the

United Kingdom, France, Bulgaria, Canada, Hungary, Spain and other countries had

been reported. The earliest occurrence of powdery mildew in China was reported by

Japanese scholar Kanji Sawada in Taiwan Province in 1919 and identified as

Erysiphe Polygoni DC. Then there were reports of tomato powdery mildew in

Xinjiang, Heilongjiang and Liaoning, but the results of pathogen identification of

tomato powdery mildew were different. In recent years, The pathogen of tomato

powdery mildew is Oidium neolycopersici was reported in Jilin, Henan, Taiwan,

Gansu and Heilongjiang provinces. Powdery mildew in tomato is a common disease

in China, but no reports were involved in the identification of powdery mildew in

tomato in the city of Yinchuan, Ningxia Hui Autonomous Region.

Therefore, this study conducted morphological and molecular biological

identification of tomato powdery mildew pathogen in Yinchuan region, in order to

provide theoretical basis and reference for the prevention and treatment of tomato

powdery mildew in Yinchuan.

Methods The pathogen of tomato powdery mildew in Yinchuan city was identified

by morphology and molecular biology. First, The collected tomato powdery mildew

leaves were brushed into sterile water containing 0.05% Tween -20 and the spore

suspension was fully shaken. Add a drop of spore suspension and observe Conidia,

Germ tube, Conidiophore, Hyphae and Appressorium under Olympus SC180

microscope. Molecular biological identification: The general primers ITS1 and ITS4

were used for PCR reaction. PCR products were detected by 1% agarose gel

electrophoresis and the target band was sequenced. Sequences using SeqMan

53

software sequencing measured signal peak figure, after get the complete sequence.

Nucleic acid sequence alignment was carried out in NCBI. Sites 18S, ITS1, 5.8S,

ITS2 and 28S were determined respectively. Then, sequences between ITS1 and ITS2

were selected for multi-sequence alignment in DNAMAN, and Mega7 was used to

construct phylogenetic tree for genetic evolution analysis.

Results Morphological identification results: the Conidia were present in solitary and

ovoid (or waist-drum). At the end of the bud tube there was a cleft petal or mastoid

appressorium, and appressorium attached to hyphae. The Conidiophore was erect and

unbranched, Furthermore, there was no closed sac shell, according to the above

characteristics is Oidium neolycopersici. Molecular biological identification

results:After PCR amplification, a single clear band appeared between 500 and 750

bp, and sequencing results showed that the length of the band was 640bp, which was

in line with the size of the target ITS sequence. In the ITS sequence with a total

length of 640bp, the sequence sites between ITS1 and ITS2 were 31-582 and the

length was 552 bp. The sequences of ITS1~ITS2 were selected for multi-sequence

comparison to construct phylogenetic tree. The results showed that Leveillula taurica

and Erysiphe polygoni were significantly different from Oidium lycopersici and

Oidium neolycopersici in base sequence and evolutionary distance, but there was no

significant difference in evolutionary distance between Oidium lycopersici and

Oidium neolycopersici. However, there were differences in the evolutionary distance

between the Oidium neolycopersici reported in France and the Oidium neolycopersici

reported in other regions. By ITS sequence alignment, the consistency with the

Oidium neolycopersici was more than 99%.

Conclusions In this study, the pathogenic bacteria of tomato powdery mildew in

Yinchuan city were identified from morphology and molecular biology. The results

show that the Conidia were present in solitary and ovoid (or waist-drum). At the end

of the bud tube there was a cleft petal or mastoid appressorium, and appressorium

attached to hyphae. The Conidiophore was erect and unbranched, furthermore, there

was no closed sac shell, according to the above characteristics is Oidium

neolycopersici, and the homology with Oidium neolycopersici was more than 99%.

The results showed that the pathogen of tomato powdery mildew in Yinchuan city

was Oidium neolycopersici.

54

Applications of Environmental Economic Policies in the Prevention

and Control of Agricultural Non-point Source Pollution in China

Hanqing Wu1; Baogui Zhang1

1 College of Land Science and Technology, China Agricultural University, Beijing, China

Objectives: To realize the green development, effective prevention and control of

agricultural non-point source pollution (ANSP) is the prerequisite.

Methods: Based on the systematic analysis of the current situation, characteristics

and relevant environmental economic policies about the prevention and control of

ANSP, the study used the case analysis to screen out the environmental economic

policies suitable for the prevention and control of ANSP, and then provided a certain

basis for decision makers.

Results: The results indicated that the source control strategy is the key to the

prevention and control of ANSP. ANSP produces negative externality of economy,

but the ecological compensation policy, a positively stimulating environmental

economic policy, which was suitable for internalizing the economic negative

externalization produced by ANSP. The environmental economic policies suitable for

the prevention and control of ANSP mainly focused on ecological compensation

policy. And the applications of producer responsibility extension policy in

agricultural film residual pollution, pesticide packaging waste and ecological

compensation policy in ANSP caused by excessive use of fertilizers and pesticides

were more feasible. The principal part of the ecological compensation policy should

be diversified, and the relationship between government and market should be

complementary, whose combination will benefit the prevention and control of ANSP.

Moreover, the ecological compensation policy and the three-product one-standard

policy, environmental tax policy and producer responsibility extension policy and

green finance-related policies (such as green security, insurance, bonds, procurement

policies, etc.) can effectively control the quality of agricultural products, the use of

fertilizers and pesticides, the recycling of residual film and pesticide packaging waste,

meanwhile, expand the ecological compensation fund pools, so as to better prevent

and control the generation of ANSP.

55

Conclusions: There are still many problems in the application of environmental

economic policies in the prevention and control of ANSP, and how to combine

ecological compensation policy with other related environmental economic policies

efficiently and how to supervise the effects of environmental economic policies still

needs further study.

Acknowledgements: This work was supported by the National Natural Science

Foundation of China (Grants No. 2016YFD0800906).

Indian River Research and Education Center, Florida ...

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