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Agricultural Sciences in China Vol.2No.10 11371143 October2003 E ffects and Mechanisms of Oil Cakes on the G rowth of Cucumber

(Cucumis sativus L.)Under Continuous Cropping System

RUAN Wei2bin1,L IU Mo2han1,PAN Jie2,Wang Jing1,L U Wen2long2,

MA Cheng2cang1,WAN G Jing2guo3,SHEN Yue4and GAO Yu2bao1 (1College of L if e Sciences,N ankai U niversity,Tianjin300071,P.R.China;2Tianjin Academy of A gricultural Sciences, Tianjin300192,P.R.China;3Depart ment of Plant N ut rition,China A gricultural U niversity,Beijing100094,

P.R.China;4A gro2Environmental Protection Institute of Minist ry of A griculture,Tianjin300191,P.R.China)

Abstract:Fxperiments on the effect of soil amendment with rape(Brassica chinensis L.)seed cake,cot2 ton(Gossypium hirsutum L.)seed or sesame(Sesamum indicum L.)seed cake on the grow th of cucumber seedlings under a continuous cropping system w ere conducted in a greenhouse environment.The results indicat2 ed that t w o applications of sesame seed cake(0.1and0.5%,w/w)increased the grow th of cucumber,but the rape oil cake show ed a neg ative effect at a rate of1.5%(w/w).The ses am e seed cake w as sep arated into four fractions (Ⅰ,Ⅱ,ⅢandⅣ)according to the polarity,and all four fractions h ad a positive effect on the grow th of cucumbers u n2 der a continuous cropping system.F raction numberⅢw as isolated into25proportions by silicon colum n,and only ole2 ic acid,p almitic acid and octadecanoic acid w ere identified in proportion10by G C2MS in w hich93.3%w as oleic acid and p almitic acid.The oleic acid h ad a significant and positive effect on cucumber grow th u nder s alt stress at the level 30mmol and show ed slight resistance to several p athogenic fu ngi.

K ey w ords:Cucumber,Oleic acid,Continuous cropping,Salt stress,Fertilization,Oil cake

During2002,5.2×108t of vegetables from1.7×107ha were produced in China.However,only0. 86%of those vegetables was exported because of their poor quality.At present,the area of greenhouse2 grown vegetables has exceeded1.2×https://www.doczj.com/doc/a912815693.html,rge2 scale cultivation,specialization,and industrialization of vegetable production in a limited area have led to the pneumonia that many kinds of vegetables were continuously cropped in the same field.This is one of the most important reasons for the reduction of the vegetablesπquality and quantity.Under this condi2 tion,both biotic factors(such as root knot nematode, Meloi dogyne arenaria,Fusari um oxysporum,and other deleterious organisms)and abiotic factors(such as s oil salinity)interact synergistically to damage vegetable growth and reduce yield.These diseases,which are referred to as“s oil2sickness”,limit the profitability and sustainabili2 ty of vegetable production in China[1,2].

Actually,the general method used to control this problem is the frequent application of pesticides and other chemical substances,which endanger the health of residents and the environment.For example,sev2 eral kinds of vegetables,such as leeks,had a high pesticide content because of polluted groundwater and pesticides,which had concentrated in organisms liv2 ing in the river estuarine zone[3].Now,some pesti2 cides have been gradually prohibited because of the possibility that they cause cancer and malformation. Therefore,it is very important to develop alternative measures or compounds that are environmentally friendly in order to control soil problems effectively. Naturally occurring compounds that are found in plants are especially desirable.

In China,a large quantity of rape plant is grown,which results in a large amount of oil cake. For example,6×106tons of rape cake are produced every year.Because of the high content of noxious chemical substances,several kinds of oil cake are

RUAN Wei2bin(19712),Associate Professor,Ph D,E2mail:caurwb@https://www.doczj.com/doc/a912815693.html,

not suitable for forage and therefore are discarded as waste.However,those noxious kinds of oil cake would be very useful if we regarded them as a fertiliz2 er and soil2improvement additive.

The application of oil cake in traditional agricul2 ture has a long history in Japan.After using oil cakes in combination with chemical fertilizers,both yield and quality of flue2cured tobacco were improved,es2 pecially the tobaccoπs fragrance and high fatty acid content[4].Soaking eggplant and tomato with an ex2 traction of castor oil cake inhibited the damage of root knot nematode and promoted the eggplantπs and tomatoπs growth[5].In recent years,a lot of oil cake has been used in the production of greenhouse vegeta2 bles,meliorating soil texture and other characters while increasing the quality of the plants.However, the effects of the oil cake on the plants differed largely from each other as a result of different compounds in them.Our objective was to study the effect of several kinds of oil cake on cucumber growth under a contin2 uous cropping system,and isolate any efficient chemi2 cal substances.At the same time research was2 ducted concerning the effect of these substances on certain fungi and its resistance to salinity.

1 Materials and Methods

1.1 The effect of oil cake on cucumber growth

Studies were conducted in soils from the Xiao2 bianzhuang,Nanhe Town,Xiqing District,Tianjin, China,in which cucumbers were planted in seven successive production cycles.In this area,the sick soil where cucumber seedlings had wilted and almost died,was sampled.The main property of the soil was available N132.8mg kg1,available K147.5mg kg1,Olsen2P536.8mg kg1,p H7.95,and the to2 tal salt content was0.223%.Soils were fertilized with N30mg kg1,P2O520mg kg1,K2O30mg kg1,and three kinds of oil cake,including B rassica chi nensis L.,Gossypi um hi rsut um L.,and Sesa2 m um i ndicum L.Oil cakes were incorporated into the soils at a rate of(w/w)0.5and0.1%for Sesa2 m um i ndicum L.,1.5and0.5%for the others.Ten cucumber seeds(Jinchun4,the same as below)were sowed on Sept.18,2001,and after germination the seedlings were thinned to3plants per pot,and finally harvested on Dec.17,2001.At harvest time,plant height,dry weight,leaf area,total root length (TRL),and other indexes were measured.

1.2 The effect of crude extracts from Se samum in2

dicum L.oil cake on cucumber growth

Sesam um i ndicum L.oil cake was extracted by 95%ethanol for8h under reflux.After evaporating the ethanol,the residue was partitioned and then di2 vided into four fractions:I(water),Ⅱ(methanol),Ⅲ(CCL4),Ⅳ(hexane).The mass of each fraction was mixed with the soil according to its proportion in the oil cake.The final ratios of fractions I,Ⅱ,ⅢandⅣ(soil,w/w)were0.07,0.07,0.2and0. 38%,respectively.Five seeds were sown on Dec.7, 2001,and after germination the seedlings were thinned to1plant per pot,and then harvested on Dec.23,2001.At harvest time,some growth pa2 rameters were measured.All of the pots were placed in a greenhouse.The growth conditions were as fol2 lows:light was provided by high pressure sodium (HPS)lamps with photosynthetic photon flux(PPF) density approximately200μmol m2s1for a photope2 riod of12h on cloudy days and4h during18:00 22:00on sunny days.The greenhouse was set at room temperature.

1.3 Chemical isolation and identification

FractionⅢwas subjected to column chromato2 graphy on silica gel60and eluted successively with CHCl32MeOH(9∶0.5),and then23proportions were obtained.The mass of proportion10was92% of fractionⅢ.The chemical composition of fraction Ⅲand proportion10was determined with gas chro2 matography2mass spectrometry(GC2MS)(HP G 1800A GCD system).The flow rate was 1.0ml min1.The carrier gas was He.The temperature was programmed to4℃every minute,from50℃to300℃. The column press was53kPa at50℃.

1.4 Re sistance determination on fungi

The bioassay of resistance on Bot rytis ci nerea was determined by cucumber cotyledon method,and prochloraz as check.For bioassay of resistance on

8311RUAN Wei2bin et al

Fusari um oxysporum f.sp.cucumeri um ,Col 2letot richum lagenari um ,

Scleroti nia sclerotiorum

and Cladospori um cucumeri num ,the method of mycelium growth rate during a 722hour period was used with deionized water as a check because of its ze 2ro resistance.All bioassays were conducted in the Na 2

tional Pesticides Engineer Research Center (Tianjin ).1.5 The re sistance of plants treated with oleic acid

on salt stre ss

The experiment consisted of the following treat 2ments:C K ,NaCl (30mmol L 1

),NaCl (30mmol L 1)+oleic acid (100μl L 1),NaCl (30mmol L 1)+oleic acid (300μl L 1),NaCl (30mmol L 1)+ole 2ic acid (500μl L 1

).Each treatment had 7repli 2cates.During the experiment ,the oleic acids were added to the solutions directly.On Oct.15,2002,seeds were germinated in vermiculite.At 7DAP (days after planting ),the seedlings πroots were rinsed cleanly and then transferred to 12L plastic pots con 2taining 1L of half 2strength C K nutrient solution.The half 2strength C K nutrient solution was replaced by a complete solution at 9DAP ,and then renewed every three days.The pots were placed in a green 2house with the following conditions :Light was pro 2vided by high pressure sodium lamps with photosyn 2thetic photon flux density at approximately 200μmol m

2

s 1

for a photo 2period of 14h at room temperature.During the experiment ,air was continuously pumped into the solution to supply oxygen.The water culture nutrient solution consisted of (mmol )0.75K 2SO 4,0.65MgSO 4?7H 2O ,2.0Ca (NO 3)2?4H 2O ,0.25KH 2PO 4,0.1ED TA 2Fe ,0.02H 3BO 3,0.001Mn 2

SO 4?H 2O ,0.0001CuSO 4?5H 2O ,0.001ZnSO 4?7H 2O ,0.1KCl and 0.000005(N H 4)6Mo 7O 24?4H 2O.On Nov.13the plants were harvested ,and some growth characteristics were measured ,including the SOD activity [6].The SOD activity unit corre 2sponded to 50%inhibition of formazan production.Peroxidase activity was determined using guaiacol as the hydrogen donor as previously reported [7].Peroxi 2dase activity was expressed as the increase absorbance at 470nm min 1g

1

FW.

1.6 Statistical analysis

All of the data were processed by SAS 6.12.Means were compared using L SD at the P <0.05probability level.

2 Re sults

2.1 Bioa ssay of oil cake

Different kinds of oil cake showed different ef 2fects on the growth of cucumber seedlings (Table 1).When the rape cake fertilizer was used to fertilize the soil at the level of 1.5%,it inhibited the growth of cucumber seedlings considerably.Seedling death was observed and the root dry weight and TRL were quite https://www.doczj.com/doc/a912815693.html,pared with C K ,at a level of 0.5%the rape cake still showed some negative effect on the plant.However ,when 0.5%cotton cake was used ,signifi 2cant stimulation resulted.The dry weight ,leaf area and total root length were all superior to that of C K.After increasing the amount of cotton seed cake to a level of 1.5%,the results of the cucumber growth were no better than when the level was at 0.5%.

T able 1 The effect of soil amendments on the grow th of cucumber under a continuous cropping system 1

)

Treatment Plant height

(cm )Shoot DW

(g pot 1)Root DW

(g pot 1)Leaf area

(cm 2)TRL

(cm pot 1)Leaf number

(number per pot )CK 13.2±1.5a 0.56±0.14c 0.06±0.02c 9.0±2.5d 1499±444bc 17.3±1.51b Rape 1.5% 5.8±2.5b 0.16±0.12d 0.01±0.01d 1.8±2.1e 311±210d 9.8±4.02c Rape 0.5%11.7±1.2a 0.63±0.16c 0.07±0.02c 12.9±1.5c 1234±326c 17.2±1.17b Cotton 1.5%13.2±3.3a 0.66±0.21c 0.07±0.03c 12.7±2.6c 1331±643c 17.0±2.28b Cotton 0.5%16.1±1.0a 0.89±0.09b 0.10±0.01b 15.9±1.1b 1732±209bc 18.3±1.03b Sesame 0.5%22.7±2.1a 1.50±0.16a 0.17±0.02a 24.1±3.6a 3220±641a 20.8±0.75a Sesame 0.1%

17.0±1.7a

0.85±0.11b

0.10±0.02b

14.3±2.4bc

1985±531b

18.8±0.75ab

1)

Means within a column followed by the same letter are not significantly different at 5%level by LSD test.The same as below

9

311E ffects and Mechanisms of Oil Cakes on the Growth of Cucumber (Cucumis sativus L.)Under Continuous

After treating with sesame cake at a level of 0.5%,the growth of cucumber seedlings improved dramati https://www.doczj.com/doc/a912815693.html,pared with the C K ,the plants πheight ,shoot dry weight ,root dry weight ,leaf area ,and TRL was 172,268,283,268,and 215%,res pec 2tively.Even when the content of sesame cake was de 2creased to 0.1%,it still positively affected the growth of the cucumber.

2.2 Bioa ssay of different fractions of se same cake

The effects on the growth of cucumber seedlings under conditions of different fractions of sesame cake mixed with sick s oil are showed in T able https://www.doczj.com/doc/a912815693.html,pared with C K ,all treatments significantly improved the growth of cucumber plants.Both root and shoot dry weight in the treatment of fraction Ⅲand fraction Ⅳwas as much as three times that of C K.Moreover ,the TRL ,leaf number ,and leaf area also increased significantly.

T able 2 E ffect of different fractions of oil cake (Sesam um indicum L.)on the grow th of cucumber seedlings under continuous

cropping system

Treatment Plant height

(cm )TRL

(cm pot 1)Leaf area

(cm 2)Shoot DW

(g pot 1)Root DW

(g pot 1)Leaf number

(number per pot )CK 5.79±1.80c 804.5±453.7c 14.7±7.9d 0.219±0.128d 0.057±0.032c 3.29±0.76b Fraction I 7.21±1.56c 1932.5±399.6b 25.6±7.8c 0.395±0.106c 0.100±0.027b 3.86±0.38a Fraction Ⅱ9.67±1.35b 2202.4±427.8b 32.8±9.6bc 0.504±0.101bc 0.124±0.021b 4.14±0.38a Fraction Ⅲ10.60±1.33b 3109.8±880.4a 36.1±8.5ab 0.610±0.167ab 0.160±0.031a 4.14±0.38a Fraction Ⅳ

12.39±1.03a

3004.6±414.6a

44.1±5.9a

0.712±0.100a

0.171±0.021a

4.00±0.00a

2.3 The isolation and identification of the active

substance s in fraction Ⅲ

There was a sharp promotion of the growth of cucumber when the sick soil was mixed with fraction

Ⅲand fraction Ⅳ,but the application weight for fraction Ⅲwas almost half of fraction Ⅳ.Thus ,fraction Ⅲwas chosen for further analysis.Fraction Ⅲwas separated by column chromatography with sil 2ica gel G60and 23proportions were collected ,in

which mass of tenth proportion is the biggest.Frac 2tion Ⅲand the tenth proportion were analyzed by GC 2MS.The result indicated that the tenth propor 2tion consisted of three pure compounds :palmitic acid 41.517%,oleic acid 51.808%,and stearic acid 6.

675%,respectively (Fig.).The contents of Fraction

Ⅲwere palmitic acid 49.026%,oleic acid 42.058%,and stearic acid 4.704%,adding up to a total proportion of 95.788%.

Fig. G C 2MS spectrum of the 10proportion of the fraction Ⅲ

0411RUAN Wei 2bin et al

2.4 The resistance of oleic acid to plant pathogenic

fungi

At high concentration,each sample shows slightly direct resistance to Bot rytis ci nerea,while showed no direct dead effect on the other pathogenic fungi(Table3).

T able3 The effects of the fractionsⅢ,Ⅳ,oleic acid and palmitic acid on several plant pathogenic fungi1)(%)

Compound

(mg L1or ml L1)Concentration

Fusari um

oxysporum f.sp.

cucumeri um

Colletot richum

lagenari um

Bot rytis

ci nerea

Scleroti nia

sclerotiorum

Cladospori um

cucumeri num

Crude extracts500022.200 FractionⅢ50015.2

5000000 FractionⅣ500 6.5

5000000 Oleic acid500047.100 5000000 Palmitic acid50033.3

5000000 1)“”:Not determined

2.5 Antagonistic effect of oleic acid under salt

stre ss condition

The analytical result of the soil showed that the experimental soil was salty soil.Under solution culti2 vation,the oleic acid was added directly to the culture so as to monitor the antagonistic effect of oleic acid on salt stress(Table4).It indicated that the root and

shoot dry weight were promoted with the increasing level of applied oleic acid in the https://www.doczj.com/doc/a912815693.html,pared with C K,the oleic acid affected the POD activity in2 significantly and reduced the SOD activity gradually with the level of oleic acid increasing.The difference was especially significant at the levels of300and500μl L1.

T able4 The effects of oleic acid on grow th and POD and SOD activity of cucumber under salt stress at30mmol L1

Treatment Shoot DW

(g pot1)

Root DW

(g pot1)

POD

OD470min1g1FW

SOD

U h1g1FW

CK 2.456±0.204a0.596±0.077a27.9±13.6b764.0±167.2c NaCl 1.730±0.243c0.294±0.032c85.8±20.1a1375.9±472.8a NaCl+100μl L1oleic 1.750±0.123c0.328±0.014c102.6±24.9a1338.4±235.0a NaCl+300μl L1oleic 1.810±0.159b0.371±0.065b104.6±33.3a1075.6±267.7b NaCl+500μl L1oleic 1.959±0.228b0.374±0.041b111.1±67.1a986.6±235.5bc

3 Discussion

The amendment of sick soil with 1.5%rape cake clearly inhibited plant growth,suggesting a tox2 ic effect from rape cake at a level of1.5%.Negative effects on the growth of tomato were reported when rape cake was added to soil at a high level[8].The growth of cucumber was greatly improved when treated with sesame cake,demonstrating that the crude extraction of oil cake contains some kinds of ac2 tive substances,which could meliorate the growth of cucumber under a continuous cropping system.Adding sesame cake and urea distinctly increased plant height,leaf area index,and the content of sug2 ar[9].

The bioassays of the four fractions obtained from the sesame cake by partition indicated that all of the fractions significantly improved the growth of cucum2 ber seedlings,which demonstrates again that the sesame cake added to the sick soil had a positive effect on the growth of cucumber.The main components of fractionⅢwere fatty acids,which outstandingly im2 proved plant growth at a relatively small quantity and therefore is important in the further practice under

1411

E ffects and Mechanisms of Oil Cakes on the Growth of Cucumber(Cucumis sativus L.)Under Continuous

the field conditions.

Our previous study also indicated that oleic acid does not directly stimulate the growth of cucumber seedlings under solution culturing.In fact,at a level of10μl L1,the oleic acid shows almost no negative effect on the growth of cucumber while the other three levels of100,500and1000μl L1show certain inhibi2 tion of the growth,respectively[10].

It was reported that plant pathogens are impor2 tant factors,which account for the yield reduction of cucumber planted in the sick soil.In the present study,we tested the fungicidal activity of several samples.The results showed that all samples had poor or no resistance to the fungi.However,we can2 not exclude the possibility of inducing the systemic resistance in plants.

The soil used in the experiment was a saline soil. The result of preliminary experiment showed that the cucumber leaves were in chlorosis and their biomass was reduced seriously even at the level of30mmol L1 NaCl.This induced the fact that the cucumber is sen2 sitive to salt stress.Increasing the oleic acid supply resulted in an increase in the root and shoot dry weight,especially at the levels of300and500μl L1. This suggested that the oleic acid was very antagonis2 tic to salt stress.Our results were consistent with the study by G ong et al.[11]and Zhao and Shu[12].Ex2 ogenous stearic acid and linoleic acid decreased Na+ absorption and transportation to the shoots,increased K+absorption and transportation,decreased the leak2 age of electrolytes,and enhanced the activities of tonoplast H+2A TPase and H+2PPase.This showed that the two fatty acids miti2gated salt stress to some extent[11].Zhao and Shu[12]demonstrated that one of the mitigative mechanisms of exogenous fatty acids on salt injury was to improve membrane phospholipid and PA contents,leading to an enhancement in mem2 brane integrity and a change in charge status of PM vesicles.This caused the activity of membrane2associ2 ated enzyme H+2A TPase to be increased and synthe2 sis of Na+/H+antiport protein was activated[12]. When treated with oleic acid,the plant SOD activity decreased significantly.We hypothesized that it re2sulted from the enhancement of membrane integrity after the oleic addition;thus we need further analy2sis of the oxygen content radical to confirm this supposi2 tion.

Root2knot nematodes(R KN,Meloi dogyne spp.)are destructive to plants with a wide distribu2 tion all over the world.They have wide host ranges and are the cause of most economic damage.Plants can be easily infected by other pathogens after the in2 vasion of R KN.Under the continuous cropping sys2 tem,the damage of R KN is more serious than under the normal rotation system,and is the main cause of yield reduction.However,we were not able observe the damage of R KN in this research.Some seed cake such as sesame cake,rape cake,and cotton cake sup2 pressed significantly the density of root2knot nema2 todes[9,13].Moreover,the essential oils of some plants reduced greatly the galling index of R KN[14].

Because the total amount of N,P,and K in sesame is about9.8%,it will increase the content of organic carbon as the carbon source for soil microor2 ganisms and meliorate the soil ecosystem when it is added to soil.

Our understanding about the mechanism of sesame cake promoting the growth of cucumber and amending soil sickness is still very limited.Whether sesame cake leads to the plantsπsystemic resistance or affects the kind and quantity of soil microorganisms needs further study.Whether pure compounds added to sick soil enhance plant growth or not is not clear ei2 ther.In addition,oleic is hydrophobic and only exists in the solutionπs surface.We are not sure if it affects the results of related experiments.

Acknowledgments

This work was jointly supported by the Ministry of Science and T echnology,China(N K BRSF Project G2000018601and2001AA247031)and the Tianjin Nat2 ural Science Foundation(013614611).We thank Mr. Chen Jianyu for the work of G C2MS.

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E ffects and Mechanisms of Oil Cakes on the Growth of Cucumber(Cucumis sativus L.)Under Continuous

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foreign exchange 外汇 sum total 总数 per capita 人均 GNP 国民生产总值 joint ventures 合资企业 net income 净收入 world investment system世界投资体系 the stock of foreign investment 外资存量 英语翻译重点词汇(2) major economy 经济大国 the stimulus, the technology and the funding 动力、技术和资金international trade and investment 国际贸易和投资 popular science 科普读物Arctic seas 北冰洋 vegetable oil 植物油 mineral oil煤油 internal combustion engine内燃机 warfare on land and sea 陆战和海战 minute sea creatures and plants 微小的海洋动植物 crude oil 原油 sedimentary rocks 沉积岩 pitch lake 沥青湖 superhighway 高速公路 living organism 生物体 applied entomology 应用昆虫学 environmental Law环保法 wildlife 野生生物

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