全 文 ：The Allelopathy of Myriophyllum Spicatum on Freshwater Algae
HU Wei , 　ZHANG Dian , 　RAM Ying-xia , 　ZHANG Ting-ting
(College of Life Science , Anhui Normal University , The Key Lab.of Biotic Envi ronment and Ecological Safety in Anhui Province ,Wuhu 241000 , China)
Abstract:To pursue an ef fective method to control f resh w ater algae , a culture solution of submerged
macrophyte Myriophyllum spicatum was tested to study its allelopathic inhibi tory ef fects on the grow th of
Microcyst is aeruginosa and Scenedesmus obl iquus with pure or mixed culture , as well as on the grow th of algae
from the pond w ater.Our results demonst rated that M .spicatum culture solution inhibited the grow th of M.
aeruginosa and S .obliquus , with M.aeruginosa being more sensit ive.M .spicatum culture solution also
induced a decrease in chlorophyll a , a reduction in algal photosynthesis intensi ty , and a depression in the ability
of alg a cells to deoxidize T TC , indicating i ts potential to inhibi t the grow th of alg ae.Our present study show ed
M .spicatum is an effective w ay to control algae-bloom.
Key words:Myriophyl lum spicatum ;Microcystis aeruginosa;Scenedesmus obliquus ;allelopathy
Classi fication No.:Q936　　Document code:A　　Paper No.:1001-2443(2011)04-0359-06
Introduction
　　Today many lakes are highly eut rophic and turbid due to increased nutrient loaded from ag ricultural ,
indust rial and domestic sources[ 1] , resulting in a gradual increase of phy toplankton biomass and formation of
w ater bloom frequent ly.To effectively utilize w ater resources and manage aquatic environment , an important
mission for scientific researchers is to take measures to control algal grow th.Submerged macrophy tes is of g reat
importance fo r the biological structure and water quality of shallow lakes[ 2] .They are believed to play a key role
in sustaining the clear w ater state by competing wi th algae for nutrients and light
[ 3]
and directly reducing
phy toplankton and periphyton grow th by allelopathy.Certain macrophytes have been reported to contain anti-
algal and anti-cyanobacterial component , which has led researchers to explo re the potential of cont rolling the
nuisance algal and cyanobacterial g row th by using the fascinating macrophy tes[ 4-5] .Among all the submerged
macrophytes studied so far , Myriophyllum spicatum has been shown the st rongest in the inhibition of
cyanobacterium M .aeruginosa grow th[ 6] .Gross et al.showed that M .spicatum can release the allelopathic
compound hydrolyzable tannin (eugeniin), and its derivatives , ellagic and gallic acids[ 7] .Nakai et al found
pyrogallic acid , (+)- catechin and fatty acids , specifically , nonanoic , cis-6-octadecenoic , and cis-9-
octadecenoic acids are all released by M .spicatum .These phenolic acids and fatty acids can significant ly inhibit
the g row th of M.aeruginosa[ 8-10] .Taken together , these f inding s point to the possibility of alg ae control via
using M .spicatum .In o rder to further study the allelopathic inhibito ry effect of M .spicatum on other alg ae as
w ell as on algae coexistence , we observed the allelopathic effects of M .spicatum on M .aeruginosa and on S .
obliquus.We also examined the ant ialgal ef fects of M .spicatum on algae in eutroph pond w ater by a short-term
laboratory experiment so as to explore the feasibility of using M .spicatum as an ef fective macrophy te to control
algae in w ater bloom.
Received date:2011-03-03
Foundation i tems:National Natural Science Foundat ion of China(30870429);The S cience and technology plan project s of Wuhu Municipality.
Biography:Hu Wei(1986-), male , born in Anqing , Anhui Province , Master candidate , mainly engaged in Algal-inhibi tion by Allelochemical;
Corresponding author:ZHANG Tingt ing(1955-), f emale , born in Dongzhi , Anhui Province , Professor.
第 34卷 4期
201 1年 7月 　　 安 徽师 范大 学 学报(自然科学版)Journal of Anhui Normal University(Natural S cience) Vol.34 No.4July .2 0 1 1
DOI :10.14182/j.cnki.1001-2443.2011.04.005
1　Materials and Methods
　　M .spicatum was collected f rom a clear pond in a suburb of Wuhu City , Anhui Province.The algae
species M .aeruginosa and S .obliquus were provided by Insti tute of Hydrobiology , the Chinese Academy of
Sciences.The sample pond w ater w as collected f rom a pond in the campus of Anhui Normal University and the
microscopic analysis of pond water samples revealed that there existed the representative genera like Microcystis ,
Scenedesmus , Chlorel la and Anabaena , w ith Microcystis sp.and Chlorel la sp.being dominating parts.
1.1　Algal culture
　　Prior to the initiation of the experiments , the single algae species(M .aeruginosa , S .obliquus)were
g row n in 250 mL sterilized flasks f illed w ith 100 mL modif ied Hoagland medium(0.1 ×)and placed in an
incubato r , using a light intensity of 4000 lx at 25±1℃ and photoperiod 12L:12D for domestic cultures for 7-
10 days.The algae from pond w ater were cultivated in the filt rated and boiled pond w ater wi th some new
modif ied Hoagland medium(0.1×)under the cultural conditions as described above.
1.2 Preparation of M.spicatum culture solution
　　M .spicatum (FW 50g)w ith all impuri ties eliminated w as acclimated and cultured in 3000 mL beaker
w ith 500 g w ashed and sterilized sand to fix it and w ith 2 liter of modif ied Hoagland medium(0.1×)under the
same condi tions as algal culture described above fo r two w eeks[ 11] .Then the culture solution of M.spicatum
was filtered wi th millipore filter(0.22μm in diameter).The filtrate w as stored for future use.
1.3　Culture of M.aeruginosa and S .obliquus in the presence of M.spicatum culture solution
　　Under aseptic condition , 36 sterilized 100 mL flasks were filled with modified Hoag land medium and alga
species in the exponential g row th phase , added with M .spicatum culture solution and adjust to the final
concentration of 0 , 10%, 20%, 40%, 60%and 80%, v/v , correspondent to 16.7 , 33.4 , 66.8 , 100.2 , 133.
6mg/L M .spicatum FW , respectively .In pure culture , each f lask w as added w ith 5mL M .aeruginosa o r S .
obliquus;in mixed cul ture , each f lask w as inoculated wi th 2.5mL M .aeruginosa and 2.5mL S .obliquus;the
final algae concentrations w ere approx imately 105 -106 cells/mL , respectively.Fo r the control , all the
components are the same except without using M.spicatum culture solution.All experiments were conducted in
triplicate.From each of the f lasks , 1mL culture sample w as collected daily for observing the inhibition of algal
g row th by counting alg a cells under Olympus microscope.The percentage inhibit ion(Iμt)of specif ic grow th rate
at certain concentration of M .spicatum culture solution w as calculated based on the fo rmula:
　　　　　　Iμt=100×(μc-μt)/μc ,μ=(lnNn-lnN0)/(tn-t0)
　　Here μc represents the mean control specific grow th rate , μt the mean specific g row th rate for the test
concentration t , N0 the number of cells/mL at time t0(beginning of the test)and Nn the measured number of
cells/mL at time tn[ 10] .
1.4　Effects of M.spicatum culture solution on the algae of the pond w ater
　　12 sterilized 100mL flasks were f illed w ith natural eut rophic pond w ater containing algae as described
above , and added M.spicatum culture solution in the final concentration of 30%, 50% and 80%, respectively.
For the control , no M .spicatum culture solut ion w as used.The experiments w ere conducted in triplicate.The
samples were cultivated under the aforementioned conditions for 14 days.A comparison of the grow th of the
o rganisms betw een the controls and treatments was obtained by quantifying the chlorophy ll a content and the
ability of alg ae cell to reduce 2 , 3 ,5-t ripheny l tet razolium chlo ride(TTC).
　　Colo rimetric method to test chlorophy ll a was reported earlier[ 12-13] .Briefly , 20 mL cultivated algae
solut ion w as used to determine the content of chlorophy ll a.After 0.2 mL MgCO3 suspension w as added to the
solut ion , the sample was centrifuged at 3 500×g , for 20min , and the sediment w as g rinded and ex tracted w ith
acetone , then the values of OD663 , OD645 and OD630 were measured.The content of chlorophyll a w as calculated
using the formula chlorophyll a(mg/L-1)=11.64(OD663)-21.16(OD645)+0.11(OD630).
360 安 徽 师 范 大 学 学 报 (自 然 科 学 版 ) 2011 年
　　TTC deoxidization method to test the reductive ability of alg ae cells w as derived from Ye et al.[ 14] .TTC ,
a w ater-soluble compound , can be reduced to t ripheny l formazan (TPF , a red-coloured compound)through the
catalysis of dehydrogenase
[ 15] .3mL 0.6%T TC was added into10mL cultivated algae solution with 10mL 0.5M
Na2HPO4-KH2PO4(pH7.4)buffer solution , ex tracted by ethyl acetate af ter 15h , follow ed by OD485
measurement.With a certain amount of TTC and some Na2S2O4 crystals , the red TTCH w ith ethy l acetate was
vibrated , ex t racted , the value of OD485 obtained , a standard curve made , and then the TPF of t reatment g roups
be calculated based on the curve.
1.5　Statistical analysis
　　All the analyses w ere triplicated.Data were recorded as mean±standard deviation and analy sed by SPSS
13.0.One-way ANOVA follow ed by LSD test w as used to test for the dif ferences between individual means.
The 0.05 level or less was selected as the point of minimum stat istical significance in every comparison.
2　Results and discussion
2.1　Effects of M.spicatum culture solution on the growth of M .aeruginosa and S.obliquus with pure
culture
　　M .spicatum culture solution inhibited the grow th of M .aeruginosa in all concentrations tested (Fig.
1a).The EC50 concentrations of M .spicatum culture solution (i.e., the concentrations of M.spicatum
culture solution that inhibits normal grow th of algae by 50%)for M .aeruginosa is 21%(35.07 mg/L FW).
At the lowest concentration(10%), M.spicatum started to inhibit the g row th of M .aeurginosa significantly
at day 3 , while the maximal inhibi tion was achieved at day 6 w ith the concentration of 80%(100%inhibition).
The concentrations of M .spicatum culture solution show ed a posi tive linear correlation w ith the percentage
inhibito ry activity
Fig 1　The growth cu rve of M .aeruginosa(a)and
S.ob liquus (b)w ith pure culture in M .spicatum
cultu re solution at indicated concent ration
Fig 2　The growth cu rves of M.aeruginosa(a)and
S.obl iquus(b)w ith mixed culture in M.spicatum
culture solution at indicated concent ration
　　As to the S.obliquus , M.spicatum culture solution showed a concentration-dependent inhibition on the its
g row th , w ith an EC50 of 47%(78.49 mg/L FW).At the low est concentration tested(10%), M .spicatum
36134卷第 4期　　　　　　　胡　威 ,章　典 , 冉颖霞 ,等:　穗花狐尾藻对淡水水华藻类的化感效应
culture solution didn t show ed significant inhibitory ef fect on S .obliquus.The maximal inhibito ry effect was
obtained wi th a concentration of 80% starting at day 2 (Fig .1b).The concentrations of M .spicatum culture
solut ion also showed a positive linear correlation w ith the percentage inhibitory activity (Fig .3).These results
indicated that M .spicatum is mo re prominent in inhibi tion on M .aeruginosa than S .obliquus;and further
suggested that M .spicatum can inhibi t the g row th of different algae by secreting allelopathic compounds , and
like many higher aquatic plants including Chara[ 1 , 16] , dif ferent alg ae have different sensibili ty to the plant.
2.2　Effect of M .spicatum culture solution on the growth of mixed culture M.aeruginosa and S.obliquus
　　As shown in Fig .2 , M .spicatum culture solution can inhibit the g row th of bo th M .aeruginosa and S .
obliquus in the mix ture.The inhibi tory ef fect on both algae is concentrat ion-dependent.Though the curve of
percentage inhibitory activity of M .aeruginosa could not be draw n because of algal death at very low
concentrations of M .spicatum culture solution), the percentage inhibitory activi ty of S .obliquus enhanced
w ith the concentrations of M .spicatum culture solution increased (Fig.3).The EC50 concentrations of M.
spicatum culture solution fo r M .aeruginosa and S .obliquus were 19% and 44%(equal to 31.73 and 75.
48mg/L M .spicatum FW), respectively.The results indicated that M .spicatum has stronger inhibition on
M .aeruginosa than on S .obliquus.The 100%of inhibition of the g row th of M.aeruginosa in mixed culture
w as achieved on the fif th day at the concentrations of upper 40% M .spicatum culture solution , making it
impossible to generate the specific grow th rates.
Fig 3　Influences of the dif ferent concen trat ion of
M.spicatum culture solution on the antialgal activity
　　While the inhibitory ef fect of M .spicatum culture solution
on M .aeruginosa was much st ronger than on S .obl iquus , the
g row th rate of M .aeruginosa was much higher than that of S .
obliquus in the control group , in w hich the average specific
g row th rates of M .aeruginosa and S .obl iquus were 0.273421
and 0.158776 , respectively.The current results are in agreement
w ith previous report (Yamamoto et al., 2005).One of the
reasons for the grow th rate of M .aeruginosa was higher than
that of S .obliquus in the control g roup might be the inter-species
competition w ith light and nutri tion.The ability of Microcystis
sp.to photosynthesize at rates higher than green alga may
facilitate the dominance of Microcyst is in w ater bodies[ 17-18] .The
o ther explanation might be the result of allelopathy betw een the
tw o species[ 19-20] .In our current study , higher grow th rate of
M .aeruginosa in the control group and higher death rate of M .aeruginosa in the t reated groups suff iciently
demonstrated that allelopathic inhibitory effect of M.spicatum on the M .aeruginosa is much stronger than
that on S .obliquus.
2.3　Effects of M.spicatum on the algae of the pond water
　　Although M .spicatum is capable of inhibi ting
both M .aeruginosa and S .obliquus in experimental
condition , we w anted to test whether it is also with
the potent ial to inhibit the alg ae grow n in pond
w ater.As such , we collected the algae f rom a pond
w ater and tested their response to M .spicatum
solut ion.chalorophyll a and TFP contents were
monitored at day ? As see from Table 1 , the M .
spicatum culture solution showed an inhibi tory ef fect
Table 1　Effect of M.spicatum culture solution on the
chlorophyll a and TPF contents of the algae in pond water
Concent rat ion of
the solution(%)
Chlorophyll a content
(ug/ L)
TPF content
(ug/mL)
0(control) 1984±105 1.35±0.12
30% 1731±39 1.17±0.09
50% 847±58a) 0.63±0.14a)
80% 307±44b) 0.26±0.08b)
　　Compared w ith cont rol group:a)p <0.05 , b)p <0.01
on the algae of the pond w ater in a concentrat ion-dependent manner , as indicated by significant decrease of the
362 安 徽 师 范 大 学 学 报 (自 然 科 学 版 ) 2011 年
chalo rophyll a and TFP contents w ith increased M .spicatum concentrat ion(compared wi th the control g roup ,
p <0.001 and p <0.01 respect ively).TTC reducing activi ty not only ref lects the total enzyme activity of cell
dehydrogenase but also is a sign of cell metabolism .Chlorophyll a is necessary for pho tosynthesis , and it
undergoes not only anabolism but also catabolism in no rmal condition in algal cell , and its synthesis is affected
w hen the grow th condition changes:when the g row ing environment is less optimal o r algal cell is suffering f rom
depression , the rate of photosynthesis w ill drop signif icantly.The results of the present study fully reveal the
allelopathic effects of M .spicatum on the algae of the pond w ater.It is the allelopathic effects of M .spicatum
that forced the chlorophyll a to be destroyed , the photosynthesis intensity decreased dramatically , and the ability
of alga cells to deoxidize t ripheny lte trazolium chloride (TTC)reduced g reatly .Overall , by inhibition of algae
g row th , M .spicatum treatment makes the w ater cleaner.
3　Conclusions
　　In this study , we found that:(1)M .spicatum culture solution can inhibit the g row th of M .aeruginosa
and S .obliquus in a concentration-dependent manner , with M .aeruginosa being more sensitive;(2)M.
spicatum culture solution can also inhibit the grow th of algae collected from the pond water , which w as reflected
in the decrease in chlorophyll a , algal photosynthesis intensity and the ability of alga cells to deoxidize T TC.Our
results f rom the current study point to the feasibility of using M.spicatum as an ef fective macrophyte to control
algae of bloom-forming.Fo r evaluation of the practical use of our finding s , further research is w arrant to study
the actual ef fect of M .spicatum on water-blooming in natural ponds o r lakes , and to observe the effects of
environmental facto rs such as w ater pH and temperature as well as nutrient limitation on the allelopathic
compounds released by M .spicatum and thei r algae g row th inhibition.
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36334卷第 4期　　　　　　　胡　威 ,章　典 , 冉颖霞 ,等:　穗花狐尾藻对淡水水华藻类的化感效应
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穗花狐尾藻对淡水水华藻类的化感效应
胡　威 , 　章　典 , 　冉颖霞 , 　张庭廷
(安徽师范大学生命科学学院 ,生物环境与生态安全安徽省重点实验室 ,安徽 芜湖　241000)
摘　要:为了寻找到一条有效治理淡水水华的途径 ,研究了大型沉水植物穗花狐尾藻的培养液对铜绿微囊藻
和斜生栅藻的单独培养及混合培养的化感抑制效应 ,同时研究了穗花狐尾藻培养液对池塘混合藻类的化感
抑制效应.结果表明:穗花狐尾藻培养液可抑制铜绿微囊藻和斜生栅藻的生长 ,对铜绿微囊藻抑制效应尤为
明显;穗花狐尾藻培养液还可导致池塘混合藻叶绿素 a含量下降 ,藻细胞还原能力降低.总之 ,该研究提示利
用穗花狐尾藻的化感作用可能是一种控制水华藻类的有效方法.
关键词:穗花狐尾藻;铜绿微囊藻;斜生栅藻;化感作用
364 安 徽 师 范 大 学 学 报 (自 然 科 学 版 ) 2011 年