Studies on the Isolation,Identification and In Vitro Growth Rates of the Three Pathogenic Fungi from Panax notoginseng Cultivated in Wenshan Eparchy

Wenya WANG,Changling ZHAO,2*,Zhongjian CHEN,Guosong WEN,2,Fugang WEI,Tingju LONG,Sunwen LI,Chongde WANG

1.College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;

2.Institute of the Improvement and Utilization of Characteristic Resource Plants,Yunnan Agricultural University,Kunming 650201,China

3.Miaoxiang Sanqi Industrial Corporation Ltd.of Wenshan City,Wenshan 663000,China;

4.Sanqi Research Institute,Wenshan University,Wenshan 663000,China;

5.Teaching Center of the Basic Experiments of Agricultural Majors,Yunnan Agricultural University,Kunming 650201,China;

6.College of Plant Protection,Yunnan Agricultural University,Kunming 650201,China

Studies on the Isolation,Identification and In Vitro Growth Rates of the Three Pathogenic Fungi from Panax notoginseng Cultivated in Wenshan Eparchy

Wenya WANG1,Changling ZHAO1,2*,Zhongjian CHEN3,4,Guosong WEN1,2,Fugang WEI3,Tingju LONG3,Sunwen LI5,Chongde WANG6

1.College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;

2.Institute of the Improvement and Utilization of Characteristic Resource Plants,Yunnan Agricultural University,Kunming 650201,China

3.Miaoxiang Sanqi Industrial Corporation Ltd.of Wenshan City,Wenshan 663000,China;

4.Sanqi Research Institute,Wenshan University,Wenshan 663000,China;

5.Teaching Center of the Basic Experiments of Agricultural Majors,Yunnan Agricultural University,Kunming 650201,China;

6.College of Plant Protection,Yunnan Agricultural University,Kunming 650201,China

[Objective]The aim of this study was to simultaneously isolate and identify the main pathogenic fungi of the root rot,black spot and round spot from thePanax notoginsengplants cultivated in Wenshan Eparchy of Yunnan Province of China.[Method]The pathogenic fungi were isolated and purified by using potato dextrose agar(PDA)medium.The morphological identification was accomplished first according to the colony forms of the fungi when cultivatedin vitro,then according to the symptom characteristics and colony forms of the re-isolated fungi in the reverse inoculation experiments.The molecular identification was performed according to the amplification and alignment of the internal transcribed space(ITS)sequences of the fungi.The increases of the diameters and thickness of the colonies of the fungi cultivatedin vitrowere employed to indicate the growth rates of the fungi.[Results]The consistency of the colony forms and symptom characteristics and the 96%-99%similarities revealed in the ITS sequence alignments all proved that the main pathogenic fungi of the root rot,black spot and round spot of theP. notoginsengplants raised in Wenshan wereCylindrocarpon didymium,Alternaria panaxandMycocentrospora acerina,respectively.When cultivatedin vitroin the same temperature,humidity and illumination,the increases of the colony diameters and thickness ofC.didymiumwere the highest,followed by those ofA.panax, then those ofM.acerina.During different cultivation periods,the differences of the colony diameters and thickness of the three fungi all reached extremely significant level.However,at the same cultivation time,the differences of the diameters and thickness among the three fungi only reached significant level.[Conclusion]The main pathogenic fungi which result in the root rot,black spot and round spot of theP.notoginsengin Wenshan areC.didymium,A.panaxandM.acerina,respectively.When these three diseases break out at the same time,the root rot will spread fastest,followed orderly by the black spot and the round spot.

Panax notoginsengcultivated in Wenshan Eparchy;Root rot,black spot and round spot;Pathogenic fungus;Growth ratein vitro

T heRadix notoginsengetRhizomaproduced in Wenshan Eparchy of Yunnan Province of China is the genuine medical material ofRadix notoginsengetRhizoma,its yield and quality are both the first in the world[1].However,root rot,black spot and round spot diseases occur frequently on thePanax notoginsengplants cultivated in Wenshan.Being caused by the fungi and bacteria which transmit via soil,the root rot is the most severe disease to harm the underground part ofP.notoginseng,

Right now,the strategy of integrated control is adopted in the control of the root rot,black spot and round spot diseases of theP.notoginsengin Wenshan.Nevertheless,due to the deficient insight into the occurring laws,particularly the pathogenic fungus types and the relative spreading rates,of the three diseases,the main control of the diseases is still the arbitrary chemical one.For example,the fungicide,e.g.Carbendazol,and bactaicide,e.g.Phenazine oxide,are often combined to control the root rot[6], the spraying of Tuzet,Ambam,Zineb, Polymyxin WP,Ningnanmycin and so on is employed to control the black spot[4,7],and Ambam,Mancozeb and so on are utilized to control the round spot[8].Despite the quick and high controlling effects,chemical control has severe side effects,e.g.killing the natural enemies,resulting in the drug resistance of the pathogenic fungi,polluting the ecological environment,remaining the reagents hazardous to human beings and livestock,lowering the quality and commercial values ofRadix notoginsengetRhizomaand so forth[9-10].So,in the future,how to carry out reasonable biological control for the root rot,black spot and round spot of theP.notoginsengin Wenshan based on the clear insight into the occurring laws of the three diseases will be one of the key issues in the disease-controlling of theP. notoginseng[2].

The pathogenic fungi of the root rot,black spot and round spot ofP. notoginsenghas been primarily researched during last three decades in order to satisfy the controlling demands.For example,one of the pathogenic fungi of the root rot was ever reported to be the ones ofCylindrocarpon[11],Alternaria panaxWhetzel was commonly thought to be the pathogenic fungus of the black spot[4,12], and the pathogenic fungus of the round spot was identified asMycocentrospora acerina(Hartig)Deighton[5]. Above all,previous researchers usually isolated and identified the pathogenic fungi of one of the three diseases alone,which was difficult to provide the systematic understanding of the pathogenic fungus types and the spreading rates of the three diseases for theP.notoginsengin Wenshan where the three diseases occurred jointly and led to the grave loss for the production ofRadix notoginsengetRhizoma.

So far,the simultaneous isolation and identification and thein vitrogrowth rates of the pathogenic fungi of the root rot,black spot and round spot of theP.notoginsengin Wenshan have not been reported.Therefore,for the first time,this paper dealt with the simultaneous isolation of the pathogenic fungi of the root rot,black spot and round spot of theP.notoginsengin Wenshan,the comprehensive morphological and molecular identification and the real time measurement of thein vitrogrowth rates of the fungi, aiming to provide a reference for the reasonable and effective control of the three diseases of theP.notoginseng.

Materials and Methods

Collection and pretreatment of plant materials

The sick plants of the root rot, black spot and round spot of theP.notoginsengin Wenshan were randomly collected from the Sanqi Sci-Tech Demonstration Garden of Miaoxiang Sanqi Industrial Corporation Ltd.which was located in Tanke Village,Panlong Country,Yanshan County,Yunnan Province(104°19′21″E,23°31′48″N).P.notoginsengplants were cultivated on the ridges which were oriented in a north and south direction,and about 1.5 m wide,30 cm high and 20-30 m long,and were covered by a black double-layered plastic sunshade net. Being supported by the cement columns which were 7 cm×7 cm thick and 1.60 m long,the net was about 1.45 m distant from the ridge surface. The typical symptom organs,i.e.the rhizomes of the root rot[11],the aerial stems of the black spot[4],and the leaves of the round spot[5],were cut off, quickly buried in ice bags,then stored at-4℃for use(Fig.1).

Isolation and identification of the main pathogenic fungi of the root rot,black spot and round spot of the P.notoginseng in Wenshan

The isolation and purification of the pathogenic fungi were carried out by using potato dextrose agar(PDA) medium[13].In order to inhibit the bacterium growth,chlormycetin and ampicillin were added into the medium in advance,and their final concentrations were both 100 μg/ml.The symptom organs were washed with tap water.A piece of pathological tissue was cut from the juncture of the pathological and healthy tissues,soaked in 70% ethanol for 30 s,then disinfected in 5% sodium hypochlorite for 3 min,finally washed thrice by ddH2O.The clean tissue was further cut into smaller pieces of about 2 mm3,then placed on the medium plate and pressed slightly, and cultivated under constant temperatures for 4-5 d.The cultivation temperature of the tissues of the root rot was 22℃[11],that of the black spot was 25℃[12],and that of the round spot was 20℃[5].Additionally,the tissues of the root rot and black spot were cultivated in darkness and that of the round spot was cultivated under illumination.After the mycelia came out,the mycelia at the edge of the colony was picked out by using a sterile dissecting needle and inoculated again on new plates for purification,the corresponding cultiva-tion temperatures were adjusted to 20[14],25[15],and 20℃[16],respectively, and the illumination conditions remained unchanged.At the 3rd,5thand 7thday of cultivation,the obverse and back sides of the colonies were photographed,and compared with the graphs and descriptions in the literatures to accomplish the first morphological identification of the fungi[4,17-19]. Subsequently,the second morphological identification was begun with the following reverse inoculation experiments.The clean and healthy rhizomes,aerial stems and leaves of one-year-oldP.notoginsengplants were cut out nicks which were about 2 mm deep.The nicks were disinfected with alcohol for 30 s,then washed thrice by ddH2O,finally inoculated respectively with the above isolated fungi and cultivated under illumination at 28,25 and 19℃,respectively.7 d later,the expected symptoms were searched and the fungi at the nicks were re-isolated,re-purified and re-identified based on the PDA medium by using above mentioned method[20].

On the other hand,by using the fungi purified from the primitive symptom organs collected from the fields, the molecular identification of the fungi was performed based on the polymorphism of the internal transcribed space (ITS)sequences of fungous rDNA[21]. The fungus DNA was extracted by using the improved CTAB method reported in Literature[22]with minor modifications.20-25 mg mycelium was placed in 1.6 ml sterile centrifuge tube and carried out the vacuum drying at 50℃ for about 1 h.500 μl extraction buffer,i. e.TE(Tris-HCl-EDTA)buffer,50 μl 10%SDS and 65 μl CTAB/NaCl solution were used.Finally,the extracted DNA was dissolved in TE buffer(1×) and stored at-80℃for use.At the same time,three primer pairs were designed by using Primer 5.0 base on the partial sequences of the ITS regions ofCylindrocarpondidymium,A.panaxandM.acerinawhich were registered in the GenBank and whose accession numbers were AY295303, FJ607183 and AY266155,respectively(Table 1),the Tms of all forward and reverse primers were 58.01℃and59.97℃,respectively.The PCR reaction systems consisted of 11 μl DNA,5 μl 10×buffer(containing Mg2+),6 μl dNTP(2.5 mM),0.8 μl Taq DNA polymerase(5 U/μl),4 μl forward primers (10 mM),4 μl reverse primers(10 mM),and 19.2 μl RNA free ddH2O which was added finally.PCRs were carried out as follows:94℃,3 min→29 cycles(94℃,40 s→55℃,40 s→72℃,1 min)→72℃,10 min.After being detected on 1%agarose gel electrophoresis(AGE),the PCR products were sequenced by BGI,and then aligned by using Blast N,producing the sequence similarities by which the molecular identification of the fungi was accomplished.

Table 1Information of the primers used to identify the main pathogenic fungi of the root rot,black spot and round spot ofPanax notoginsengcultivated in Wenshan Eparchy by using rDNA ITS

Table 2Main pathogenic fungi identified from the symptom organs of the root rot,black spot and round spot ofPanax notoginsengin Wenshan

Determination of the in vitro growth rates of the main pathogenic fungi of the root rot,black spot and round spot of the P.notoginseng in Wenshan

The colony blocks of 1 cm2were cut out by using a sterile puncher from the purified colonies of the pathogenic fungi of the root rot,black spot and round spot,inoculated on the PDA plates and cultivated at 22℃for 7 d. At the 3rd,5thand 7thday of cultivation, the diameters and thickness of the colonies were measured by using a sliding caliper and a transparent plastic ruler,respectively.

Statistical analysis

The isolation of the fungi was repeated 4 times(5 dishes per time). The reverse inoculation experiment and the measurement of the diameters and thickness of the colonies were both repeated thrice.Every measurement of the diameters and thickness was carried out on 5 colonies.Oneway analyses of variance(ANOVA) were performed by using SPSS 17.0 and the differences were considered significant atP＜0.05.

Results and Analysis

Morphological identification results of the main pathogenic fungi of the root rot,black spot and round spot of the P.notoginseng in Wenshan Identification results based on the colony forms of the fungi cultivated in vitroFirstly,the symptom rhizomes used to isolated the main pathogenic fungi of the root rot displayed the typical“yellow rot”[11].When the pathogenic fungus of the root rot was cultivated on the PDA plate for 3 d,the colonies consisted of white and felty mycelia and the back sides of the colonies were white.When cultivated for 5 d,slight yellow hues appeared at the colony centers,but the surrounding mycelia were still white and fluffy and the back sides of the colonies became light yellow brown from the centers.When cultivated for 7 d,the colonies were composed of loose, fluffy and slight yellow brown mycelia, the yellow brown hues at the centers were much thicker,and the yellowbrown hues on the back sides of the colonies spread obviously toward the circumferences.However,during the cultivating process,the mycelia were dilute all the time(Fig.2(1)).As such, based on the comparison of above morphological changes of the fungus with those recorded in Literature[17,19], the pathogenic fungus of the root rot was preliminarily identified asC. didymium(Table 2),which was consistent with the result reported in Literature[11].

Secondly,when the pathogenic fungus of the black spot was cultivated on the PDA plates for 3 d,white villusshaped mycelia emerged from the inoculation sites,and the back sides of the colonies were white,being accompanied by yellow brown spots at the centers.When cultivated for 5 d,the colony centers became grayish black, but the surrounding mycelia were still white,and the internals of the back sides of the colonies were greenish black.When cultivated for 7 d,the grayish white centers of the colonies swelled,the surrounding mycelia were grayish black,the grayish black particle-shaped spores were observed to come out,and the internals of the back sides of the colonies were dark black, and surrounded by gray circumferences.So,the pathogenic fungus of the black spot was preliminarily identified asA.panaxof Pezizomycotina (Table 2)[4,18].Similarly,A.panaxwas confirmed to result in the black spot ofP.quinquefolium.However,when cultivated on the PDA plate,the colonies of theA.panaxfromP.quinquefoliumchanged gradually from gray white to dark green[23].

Finally,when the pathogenic fungus of the round spot was cultivated on the PDA plates for 3 d,the colony centers were light pink,surrounded by white and loose mycelia,and the back sides of the colonies became red from the centers.When cultivated for 5 d, the outmost circumferences of the white colonies were red,and the light pink centers of the back sides of the colonies were orderly surrounded by black and red concentric circles.When cultivated for 7 d,the colony centers became gray black,the outmost circumferences were still red,there were about 4 wheel-shaped streaks on the colony surfaces,and the centers of the back sides of the colonies were dark black,surrounded by redish brown circumferences(Fig.2(2)).As a result,the pathogenic fungus of the round spot was preliminarily identified asM.acerinaof Deuteromycotina (Table 2)[5,16,24-25].Furthermore,during the cultivation,it was a red and watersoluble pigment synthesized by the mycelia that made partial mycelia become red[25-26].Correspondingly,under the same cultivation conditions,the colonies of theM.acerinawhich led to the rot disease ofCoriandrum sativumwere colorless,then turned into purplish red,finally into black[27].

Identification results based on the symptom characteristics and colony forms of the re-isolated fungi in the reverse inoculation experimentBeing respectively cultivated under illumination at 28,25 and 19℃for 7 d,the excised healthy rhizomes, aerial stems and leaves of one-yearoldP.notoginsengplants inoculated respectively with the pathogenic fungi of the root rot,black spot and round spot ofP.notoginsengin Wenshan displayed the typical symptoms of the three diseases.In detail,the rhizomes showed rot(Fig.3A)[6,11],depressed spots with blackish brown mould-shaped things appeared on the aerial stems(Fig.3 B)[1,4,7,12,18], and brown round spots with wheelshaped streaks emerged and spread on the leaves(Fig.3 C)[8,24].On the other hand,when cultivatedin vitrofor 7 d,the colonies of the fungi isolated from the excised rhizomes,aerial stems and leaves all displayed the same forms as those of the first isolated fungi(Fig.2 and Fig.4).Thus,it was proven again in the reverse inoculation experiments thatC.didymium,A.panaxandM.acerinawere the main pathogenic fungi of the root rot, black spot and round spot ofP.notoginsengin Wenshan,respectively.

Molecular identification results of the main pathogenic fungi of the root rot,black spot and round spot of theP.notoginsengin Wenshan

On the agarose electropherogram,the three PCR products which were based on the DNAs of the pathogenic fungi of the root rot,black spot and round spot of theP.notoginsengin Wenshan and on the rDNA ITS sequence-specific primer pairs of the three fungi expressed three clear and bright bands whose sizes accorded with the predicted ones,and no primer dimer bands were found(Table 1 and Fig.5),indicating that the rDNA ITS sequences of the three fungi had been specifically amplified.

After being sequenced,the three PCR products were aligned respectively with the rDNA ITS sequences ofC.didymium,A.panaxandM.acerinawhich were registered in the GenBank, producing 96%,99%and 98%similarities,respectively(Fig.6).Accordingly, it was once again proven by the analysis of the polymorphism of the fungous rDNA ITS sequence thatC.didymium,A.panaxandM.acerinawere the main pathogenic fungi of the root rot, black spot and round spot ofP.notoginsengin Wenshan,respectively.

Growth rates of the main pathogenic fungi of the root rot, black spot and round spot of theP.notoginsengin Wenshan when cultivatedin vitro

Under the same cultivation condition and cultivated for the same time length,the diameters and thickness of the colonies of the three fungi, i.e.C.didymiuminducing the root rot,A.panaxinducing the black spot andM.acerinainducing the round spot, all increased persistently,and the increases of the diameters and thickness ofC.didymiumwere the quickest,next were those ofA.panax, and those ofM.acerinawere the slowest.For example,when cultivated for 7 d,the diameter of the colonies ofC.didymiumwas 9.00 cm which was about 1.13 fold of that ofA.panaxand 1.50 fold of that ofM.acerina.In the meantime,the thickness of the colonies ofC.didymiumwas 7.00 cm which was about 1.40 fold of that ofA. panaxand 1.75 fold of that ofM.acerina(Fig.7).Statistically,for the differences of the diameters and thickness of the colonies of the same fungus at different cultivation time,inF-test,F (diameter)andF(thickness)were 32.24 and 32.78,respectively,and both＞F0.01(2,4)=18,showing that,during different cultivation periods,the differences of the colony diameters and thickness of the three fungi all reached extremely significant level.In contrast,for the differences of the diameters and thickness of the colonies of the different fungi at the same cultivation time,inF-test,F(diameter)andF(thickness)were 9.96 and 6.10,respectively,and both＜F0.01(2,4)=18, but＞F0.05(2,4)=6.94,showing that,at the same cultivation time,the differences of the diameters and thickness among the three fungi only reached significant level.

Discussion

Root rot,black spot and round spot are the main diseases which occur frequently on theP.notoginsengplants raised in Wenshan and produced severe harm to the quality and yield ofRadix notoginsengetRhizoma.In this study,based on the consistency of the colony forms and symptom characteristics and the similarities revealed in the ITS sequence alignments,the main pathogenic fungi of the root rot,black spot and round spot were identified asC.didymium,A.panaxandM.acerina,respectively (Table 2).Specially,it was believed that the root rot ofP.notoginsengresulted from the comprehensive function of diverse pathogens which mainly involvedC.destructans,C.didymium,Fusarium solanin,F.oxysporum,Phytophthora cactorum,Phoma herbarum,Rhizoctonia solaniand so on,andC.destructansandC.didymiumwere the most important[11].Rhabditis axeiwas observed to promote the root rot, too[28].Moreover,the root rot was also tightly related to the moulds,actinomycetes and anoxybiontic bacteria in the rhizospheric soils[29].Above all,the pathogen types isolated from the rotted roots varied with the habitats ofP.notoginseng,rot types,occurring time of the rot,the interaction of the pathogenic germs and so on[11,30-31].In this study,the main pathogenic fungi isolated from the rotted roots wasC.didymium,which was probably because the rot belonged to“yellow”one[11].On the other hand,the main pathogenic fungi of the black spot and round spot were identified asA.panaxandM.acerina,respectively,which accorded totally with the previous research results[4,5,15-16].When cultivated on PDA plates in the same temperature,humidity and illumination,the increases of the colony diameters and thickness ofC.didymiumwere the highest,followed by those ofA.panax, then those ofM.acerina(Fig.7),which implied that,when the root rot,black spot and round spot occurred on theP.notoginseng,the root rot would spread fastest,followed orderly by theblack spot and the round spot.Thus, root rot should be regarded as the control focus in the disease-controlling of theP.notoginsengin Wenshan.

When PDA medium was used to isolate and purify pathogenic fungi,the fungi were usually polluted by miscellaneous bacteria,directly resulting in the failure of the fungi-isolating or purifying.It was found in this study that,if the concentrations of the chloramphenicol,ampicillin or“chloramphenicol+ampicillin(1:1,m/m)”which were added into the medium were all 100 ug/ml,the incidence of the miscellaneous bacteria at the 5thday of cultivation was 1/3,1/3 and 1/10,respectively.As a result,chloramphenicol and ampicillin could be both used to inhibit the pollution of the miscellaneous bacteria and the equivalent combination of chloramphenicol and ampicillin could result in the best inhibition effect. Accordingly,in the following isolation and purification of the pathogenic fungi,chloramphenicol and ampicillin were equally added into the medium and the terminal concentrations of the two antibiotics were all 100 μg/ml.

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Responsible editor:Xiaoxue WANG

Responsible proofreader:Xiaoyan WU

文山三七三种病原真菌的分离、鉴定及其体外生长速率的研究

王文亚1，赵昶灵1，2*，陈中坚3，4，文国松1，2，魏富刚3，龙廷菊3，李孙文5，王崇德6
（1.云南农业大学农学与生物技术学院，云南昆明650201；2.云南农业大学特色资源植物改良与利用研究所，云南昆明650201；3.文山市苗乡三七实业有限公司，云南文山663000；4.文山学院三七研究院，云南文山663000；5.云南农业大学农科专业基础实验教学中心，云南昆明650201；6.云南农业大学植物保护学院，云南昆明650201）

[目的]分离、鉴定文山三七根腐病、黑斑病和圆斑病的主要病原真菌，并探明其体外生长速率。[方法]用马铃薯葡聚糖琼脂培养基分离、纯化病原真菌，以真菌体外培养时的菌落形态和回接试验中的病兆特征和再分离真菌的菌落形态进行形态鉴定，以真菌ⅠTS序列扩增与比对进行分子鉴定，用体外培养时病原真菌菌落直径和厚度的增加速率来表征其生长速率。[结果]文山三七根腐病、黑斑病和圆斑病的主要病原真菌分别为双孢柱孢、人参链格孢和槭菌刺孢。体外培养时，双孢柱孢菌落的直径和厚度增大最快，其次为人参链格孢的，槭菌刺孢的则最慢；在同一培养时间，不同真菌的菌落直径、厚度间的各自差异仅达到显著水平。[结论]文山三七根腐病、黑斑病和圆斑病的主要病原真菌分别为双孢柱孢、人参链格孢和槭菌刺孢，当三种病害同时发生时，根腐病将蔓延最快，黑斑病次之，圆斑病则最慢。

文山三七；根腐病、黑斑病和圆斑病；病原真菌；体外生长速率and it is also the destructive disease ofP.notoginseng[2].Root rot usually makes theP.notoginsengyield lose about 5%-20%,and the severe rot can result in the yield loss of 70%. Moreover,this disease makes the total saponin content of the rhizomes decrease about 30.88%,and the more serious the root rot the lower the saponin content[3].The black spot belongs to fungous diseases,its incidence onP.notoginsengis about 20%-35%,and can even be more than 90%.The severely harmed organs ofP.notoginsenginclude the aerial stems,leaves,and the tender parts of the floral axes and fruit stalks[4].Recently,the black spot has been the main reason which causes the dramatic decrease of the quality and output ofRadix notoginsengetRhizomaandP.notoginsengseeds. Being a fungous disease,the round spot is a new disease which emerges and causes comparatively severe hazards in theP.notoginsengplanted area in Wenshan in recent years.It occurs mainly on the leaves,and weakens the leaf photosynthesis to impair the growth and development ofP. notoginseng[5].

国家自然科学基金资助项目（31060045，31260091，31460065）。

王文亚（1988-），女，云南曲靖人，在读硕士生，从事植物次生产物生态生理功能的研究，E-mail：461286063@qq.com。*通讯作者。

2015-04-03

修回日期 2015-05-15

Supported by the National Natural Science Foundation of China(31060045,31260091, 31460065).

*Corresponding author.E-mail:zhaoplumblossom7@163.com

Received:April 3,2015 Accepted:May 15,2015