全 文 ：Received Date:2005-05-26
＊ Corresponding Author.
Artical ID:1002-4026(2005)03-0091-07
Growth promotion of wheat seedlings
by Penicillium oxalicum
Aili.Tureke1 , 2 ,3 ,ZHAO Zhen-yu2 ,WANG Qi3 ,TANG Wen-hua1, 3＊
(1.Dept.Plant Pathology , China Agricultural University , Beijing 100094 , P.R.China;2.Dept.Plant Pathology , Xinjiang
Agricultural University , Urumqi 830052 , P.R.China;3.Beijing-Gottingen Biotech CO , Beijing 100081 , P.R.China)
Abstract:More than 100 isolates of Penicillium oxalicum were isolated from soil and roots of wheat.Among them , 5 isolates were selected for
testing the efficacy in promotion of growth of wheat seedlings.The testing was conducted in greenhouse for four times , i.e.four experiments.
The data showed that the highness of wheat seedling responded to treatment of Po-41 in three experiments was higher than CK.Fresh weight of
seedling and fresh weight of root responded to Po-41 were higher in one experiment separately comparing CK.All the difference mentioned
above was significant based on statistic analysis.Responding to treatment with Po-41 in four experiments , all indexes seemed better comparing
with that in non-treated CK , although it was not supported by statistic analysis.Analysis of phytohormones concentration in fermented solution
of the isolate showed that IAA content was high in the acetic condition.
Key words:Penicillium oxalicum;PGPR;wheat , phosphate-dissolving fungi
CLC number:　S476　　　Document code:　A
Introduction
Since 1980 s , Kucey reviewed the literatures related to role of phosphate-dissolving microorganisms.It was reported
Penicillium spp.played role in dissolving phosphorus binded on soil[ 1, 2] .Torry et al.reported mechanism of phosphorus-
dissolving action in 1993
[ 3 , 4] ;Vassilev et al , studied on role in phosphate-dissolving by Penicillium variabile in 1996[ 5] ;
Whitelaw et al , reported phosphate solubilization by Penicillium radicum in 1999[ 6] ;Reyes et al.used UV-induced
mutants of Penicillium rugulosum for increasing ability of wild type of Penicillium rugulosum in 1999
[ 7] .
Another research aspect dialed with biocontrol of plant diseases though Penicillium oxalicum.Windels reported pea
seeds treated by Penicillium oxalicum for control of manifold root wilt disease;A.De Cal et al reported that method of
application of Penicillium oxalicum to control Fusarium wilt of tomato in 1999
[ 8] ;Garcia et al.reported induce
resistance was the mechanism in control of plant disease by Penicillium oxalicum in 1998
[ 9] ;Pascual et al.studied on
environment influence on effect of controlling plant diseases by Penicillium oxalicum in 1997
[ 10] .
1　Materials and Methods
1.1　Plant material
Wheat cultivars:Zhongmai 9 was bought from the Institute of Crop in Chinese Academy of Agricultural Sciences.
The seeds used in experiments were disinfected by 0.1% sodium hypochlorite solution for 5 minutes , then washed and
kept in Petri dishes with water for 12 hours.
第 18 卷　第 3期
2005 年8 月
山 东 科 学
SHANDONG SCIENCE
Vol.18　No.3
Aug.2005
1.2　Penicillium oxalicum inoculum
1.2.1　Isolation
The soil and wheat roots were collected from wheat field in Beijing , Qinhuangdao and CAAS.Isolation was made by
planting the dis-infection roots and diluted soil on PDA with 1% Ca3PO3 , keeping it at 27℃ for 8d.Selecting
Penicillium isolates , which dissolved Ca3PO 3 .
1.2.2　Penicillium oxalicum isolates , which were tested in the experiment:Po-40 , Po-41 , Po-46 , Po-47 and BGP-01.
1.2.3　Preparation of isolates inoculation
Solid agent:The isolates of Penicillium oxalicum stored on potato dextrose agar (PDA)slants at 4℃ were
inoculated on potato dextrose agar (PDA)for spore production.As soon as the spores produced , collecting spores and
inoculating rice for producing solid agent at 27.3℃ and RH 80-90%.
Liquid solution of Penicillium oxalicum isolates.
Penicillium oxalicum isolates were grown in 250ml Erlenmeyer flasks with 150ml of PD solution at 20-25℃ on a
shaker with 150rpm for 3 days.The fermented solution was used in the experiments.
1.3 　Pot experiments on effectiveness of Penicillium oxalicum isolates on growth of wheat seedlings in
greenhouse
1.3.1　Soil:The soil used in the experiment was sterilized.Among four experiments , the soil was amendment by
phosphorite powder in three of them.One of them was not.
1.3.2　Design:In the experiment , 5 isolates were endured the pot testing under two soil conditions:2g phosphorite
powder was added into one pot(8×8×9cm)contained 200g sterilized soil in three experiments.In one experiment , the
soil was not amendment with phosphorite powder.No phosphorite powder amendment soil was settled as CK in all
experiments as CK.Each isolate was tested third times in phosphorite powder added condition.One time in the condition
of non-added phosphorite powder condition.Ten pots were settled for each isolate in one experiment.Five seeds were
planted in one pot.Seeds of wheat were dipped in spore solution of each isolate with 108 conidia ml-1 .The experiments
lasted 27 days.Highness , fresh weight , dry weight and fresh root weight of wheat seedlings were recorded.
Data analysis:All data were analyzed using analysis of variance[ 11] .Means were compared by Student-Newman-keul
s multiple range tests
[ 12] .While the difference is significant at P=0.05 in F-test , means were compared by Least Signif-
icant Difference(LSD)test.
1.4　Assays of phytohormones produced by Po-41
ELISA method was employed to determine kinds and quantity of phytohormones produced by Po-41.
Preparation of sample for assay:Taking 5ml liquid fermented solution of Penicillium oxalicum Po-41 into tubes and
adjusted pH to about 3.0.After adding 5ml acetic ether in the solution , the tubers were kept for five minutes.Then , the
same method was used to adding acetic ether until total amount of acetic ether to 13ml.Rotary evaporation of acetic ether
was made.The remainder was determined for phytohormones in acid condition.
Taking 5ml liquid fermentation solution of Penicillium oxalicum Po-41 into tubes and adjusting pH to about 10.0 ,
then adding 5ml N-dutanor ,m , depositing for five minutes.Repeating the procedure until total amount of N-dutanor was
13ml.Rotary evaporation was made.The remainder was determined in alkaline condition.
The samples , which used for further assay , were submitted to Plant Physiology Laboratory in China Agricultural
University for determination.The results listed in fig.1 ,2 ,3.
2　Results
2.1　Effect of Penicillium oxalicum isolates on growth of wheat seedlings
The data collected from experiment , in which phosphorite powder was added into soil , are putted into Table one ,
92 山　东　科　学　　　　　　　　　　　　　　　　　　　2005 年
Two and Three.The data in table one showed that the highness of wheat seedlings responsible to seeds coating by
Penicillium oxalicum Po-41 was higher then non-treated CK and treatment P , in which phosphorite powder added.The
difference was significant based on statistic analysis at level P0.01 for isolate Po-41and Po-46.However , the data in
table four showed there was not significant difference between treated and non-treated groups on highness of seedlings ,
although treatment by isolates Po-40 and Po-41 trended to be beneficial to the highness of seedlings under no phosphorite
powder added.
Table 1　Highness of Wheat Seedlings Responsible to Seed Treatment by Isolates of Penicillium
oxalicum under the condition of phosphorite powder added into Soil.
Treatment Mean N P0.05 P0.01
Po-41 30.45 37 A a
Po-46 29.71 36 A B ab
Po-47 28.66 40 A BC abc
BGP-01 27.59 40 BCD abcd
P 26.61 47 CDE bcd
CK 25.62 41 DE cd
Po-40 24.51 34 E d
Notes:P represented a treatment , in which phosphorite powder was added into soil.CK represented the treatment , in which no phosphorite
powder added , no seed treatment.The data were collected from tree experiments and analyzed.N represented numbers of wheat
seedlings.The experiment lasted for 27 days.
The data in Table two showed that there were no significant difference between treatments of Penicillium oxalicum
isolates and P.However , there was significant difference between treatment Po-47 and CK , in which the soil was not
added phosphorite powder.
Table 2　Fresh Weight of Wheat Seedlings Responsible to Seed Treatment by Isolates of
Penicillium oxalicum under the condition of phosphorite powder added into Soil.
Treatment Mean N P0.05 P0.01
Po-47 0.66543 10 A a
Po-41 0.65147 10 AB a
Po-40 0.64412 10 ABC a
P 0.61402 10 ABC a
BGP01 0.61273 10 ABC a
CK 0.55285 10 BC a
Po-46 0.53847 10 C a
Notes:N was total number of wheat seedlings.P was CK adding phosphorite powder.Different characters represented difference of statistic
analyses.The experiment lasted for 27 day s.
The data in Table three showed that there were not significant difference of dried weight between treatments and CK
and P treatment.
93第 3 期 Aili.Tureke , et al:Growth Promotion of Wheat Seedlings by Penicillium oxalicum
Table 3　Dry Weight of Wheat Seedlings Responsible to Seed Treatment by Isolates of
Penicillium oxalicum under the condition of phosphorite powder added into Soil.s
Treatment Mean N P0.05 P0.01
BGP01 0.065933 10 A a
Po-41 0.065667 10 A a
Po-40 0.064983 10 A a
Po-7 0.063283 10 A a
CK 0.061950 10 AB a
P 0.061183 10 AB a
Po-46 0.053883 10 B a
Notes:N was total number of wheat seedlings.P was CK adding phosphorite powder.Different characters represented difference of statistic
analyses.The experiment lasted for 27 day s.
At the condition of soil without phosphorite powder added , highness of wheat seedling had no significant difference
between treated by isolates of Penicillium oxalicum and non-treated control , except Po-47 treatment , in which the
response to treatment was negative.
Table 4　Highness of Wheat Seedlings Responsible to Seed Treatment by Isolates of
Penicillium oxalicum under the condition without phosphorite powder added.
Treatment Mean N P0.05 P0.01
Po-40 30.636 44 A a
Po-41 30.541 49 A a
CK 29.609 45 A a
Po-46 29.486 44 A a
BGP01 28.926 42 A a
Po-47 25.376 42 B b
Notes:N was total number of wheat seedlings.CK represented non-treated control.Different characters represented difference of statistic
analyses.The experiment lasted for 27 days.
The data in table five showed that fresh weight of wheat seedlings had significant difference between of treatment.
Response to treatment of Po-40and Po-41 , the fresh weight of wheat seedlings was significant higher than non-treated
control at level P=0.05.
Table 5　Fresh Weight of Wheat Seedlings Responsible to Seed Treatment by Isolates of
Penicillium oxalicum under the condition without phosphorite powder added.
Treatment Mean N P0.05 P0.01
Po-40 0.65693 10 A a
Po-41 0.63870 10 AB a
BGP01 0.53848 10 BC a
Po-46 0.52830 10 C a
Po-47 0.52003 10 C a
CK 0.51697 10 C a
Notes:N was total number of wheat seedlings.Different characters represented difference of statistic analyses.The experiment lasted for 27 days.
CK represented non-treated control.
94 山　东　科　学　　　　　　　　　　　　　　　　　　　2005 年
No significant difference was found in dried weight of wheat seedlings responded to treatments and non-treated CK.
The data were in the table six.
Table 6　Dried Weight of Wheat Seedlings Responsible to Seed Treatment by Isolates of
Penicillium oxalicum under the condition without phosphorite powder added.
Treatment Mean N P0.05 P0.01
Po-40 0.070967 10 A a
Po-41 0.067600 10 AB a
BGP01 0.064950 10 ABC a
CK 0.061217 10 ABC a
Po-47 0.059400 10 BC a
Po-46 0.056667 10 C a
Notes:CK represented non-treated control.N was total number of wheat seedlings.Different characters represented difference of statistic analy-
ses.The experiment lasted for 27 days.
In table seven , just two data of treatments listed including Po-41 and non-treated CK.It showed that there was sig-
nificant difference of fresh weight of root responded to treatment by Penicillium oxalicum Po-41 at level P0.05.
Table 7　Fresh Weight of Roots of Wheat Seedlings Responsible to Seed Treatment by Isolates of
Penicillium oxalicum under the condition without phosphorite powder added
Treatment Mean N P0.05 P0.01
Po-41 5.158 6 A a
CK 4.114 6 B a
Fig .1 Concentration of phytohormones concentration
produced by P-o-41 under different condition
2.2　Assays of hormones produced by Po-41
ELISA method was used for hormones determination of Penicillium
oxalicum(Po-41)under acetic and alkaline conditions.There was more
indoor acetic acid(IAA)being found in fermented solution of f P-o-41
under acid condition.
The fig 2 and 3 showed that Po-41 produced more phytohormones than
24-14 , a bacterial isolate under acid and alkaline condition.
Fig.2 Comparing the content of plant hormones produced byPo-41
and 24-14 , which is a bacterial solate under acid condition.
Fig.3 Comparing the concentration of phytohormones produced
by Po-41 and a bacterial isolate under alkaline condition.
95第 3 期 Aili.Tureke , et al:Growth Promotion of Wheat Seedlings by Penicillium oxalicum
3　Discussion and conclusion
Purpose of this study was to identify the effect of Penicillium oxalicum isolates on growth of wheat seedlings , but not
for control of diseases.Isolate BGP01 had been tested for control of seedling diseases of cotton and the role in
dissolving-phosphate[ 13] .
After preliminary selection of candidates(from more then 100 isolates)for the experiments , 5 isolates were choice
for this testing.The data listed in table 1 , 2 , 3 obtained from three experiments , in which phosphorite powderwas added
in soil.Treatment P was CK with the powder added , but the wheat seeds were not treated by any Penicillium isolates.
The data in table 4 , 5 , 6 came from one experiment(last one), in which no phosphorite powder was added.Temperature
in greenhouse was increasing from first experiment to the fourth one.
Seed treatment with isolate Po-41 showed significant difference in fresh weight of root in table 7 , fresh weight in
table 5 and highness of seedlings in table 1 comparing with CK(blank)and P (phosphorite powder).In other 4 indexes
in 4 experiments , the trend of response to treatment of this isolate was positive as well comparing with CK and P ,
although no significant difference being found.It can be recognized that the isolate Po-41 has ability to promote growth of
wheat seedlings.
Regarding to function of enhancing growth of wheat seedlings by the isolate , the experiments focused on two aspects:
dissolving phosphate binned on soil and phosphorite powder , and phytohormones.Root disease was not factor because the
soil was sterilized.There was no significant evidence to show that the isolate dissolved binned phosphate because no
significant difference was found between phosphorite powder added and not added.However , this was not final conclusion
because the period of the experiments was too short.Further research on the mechanism is needed.
Considering the role of phytohormones , the experiment showed that the isolate produced IAA and other
phytohormones.Under acidic condition , the concentration of IAA produced by the isolate may play role in promoting
growth of seedlings because the fungus produced organic acids as well.However , this point should be supported by
further experimental evidence.
With regard to reducing disease index by the isolate , new information will be published.
References:
[ 1] 　Kucey R M N , Janzen ,HH , Leggett M E.Microbially mediated increases in plant-available phosphorus[ J] .Advances in Agronomy.1989
(42):199-228.
[ 2] 　James E Cunningham , Cathy Kuiack.Production of Citric and Oxalic Acids and Solubilization of Calcium Phosphate by Penicillium bilaii
[ J] .Applied and Environmental Microbiology.1992(58):1451-1458.
[ 3] 　Angeles de la Torre et al.Biochemical mechanisms of stone alteration carried out by filamentous fungi lacing in monuments[ J] .
Biogeochemistry.1993(19):129-147.
[ 4] 　De La Torre M A , Gomez-Alarcon G Vizcaino , C Garcia M.Biochemical mechanisms of stone alteration carried out by filamentous fungi
living in monuments[ J] .Biogeochemistry , 1993(19):129-147.
[ 5] 　Vassilev N Fenice ,M federici F.Rock Phosphate solubilization with gluconic acid produced by immobilized Penicillium variabile P16[ J] .
Biotechnology , Techniques , 1996(10):585-588.
[ 6] 　Whitelaw M A Harden , T J Heylar K R.Phosphate solubilization in solution culture by the soil fungus Penicillium radicum[ J] .Soil
Biology &Biochemistry.1999(31):655-665.
96 山　东　科　学　　　　　　　　　　　　　　　　　　　2005 年
[ 7] 　Reyes I , Bernier L , Simard R R, Antoun H.Effect of nitrogen source on the solubilization of different Inorganic phosphates by an isolate of
Penicillium rugulosum and two UV-induced mutants[ J] .FEMS Microbiology Ecology.1999(28):281-290.
[ 8] 　A De Cal Garcia-Lepe , S Pascual , P Melgarejo.Effects of timing and method of application of Penicillium oxalicum on efficacy and
duration of control of fusarium wilt of tomato[ J] .Plant Pathology.1999(48):260-266.
[ 9] 　Garcia et al.Induced resistance against Fusarium wilt of tomato by Penicillium oxalicum is not associated to pathogenesis related protein
[ J] .Bulletin-Oilb Srop.1998(9):123-127.
[ 10] 　A De Cal S PASCUAL , P Melgarejo.A rapid laboratory method for assessing the biological control petential of Penicillium oxalicum
against Fusarium wilt of tomato[ J] .Plant Pathology.1997(46):699-707.
[ 11] 　Sokal RR , Rohlf FJ.Biometry[ M] .New York:W.H.Freeman and Company , 1981.
[ 12] 　Snedecor G W , Cochram W G.Statistical Methods.7th ed[ M] .Ames:Iowa State University Press.1980.
[ 13] 　Tang Wenham , et al.Effectiveness of Penicillium oxalicum-phasphate-disolving fungus on the control of damping-off of cotton caused by
Rhizoctonia solani[ A] .Proceedings of the 15th International Plant Protection Congress[ C] .2004.157.
草酸青霉对小麦苗的促生效果
艾力·吐热克1, 2 , 3 ,赵振宇2 ,王祺3 ,唐文华1, 3＊
(1.中国农业大学植物病理系 , 北京 100094;2.新疆农业大学植物病理系 ,
乌鲁木齐 830052;3.北京-高廷根生物技术有限公司 , 北京 100081)
摘要:从土壤和小麦根部分离到 100 多个草酸青霉菌 , 从中选出 5个分离物 , 测定其对小麦苗的促生长作用。在温室进行 4 次
试验。试验结果表明 ,在 3 次试验中 ,与对照相比 , 菌株 Po-41 处理组麦苗高 , 并有统计上的显著差异。 Po-41 处理还在一个实
验中 ,表现为提高幼苗鲜重 , 在另一实验中 ,提高根鲜重 ,并均有显著性差异。在 4 个试验中 , Po-41 处理组的所用其他指标 , 也
都表现比对照好 ,不过 , 没有显著性差异。 Po-41 发酵液中植物激素种类及含量测定表明 ,在酸性条件下 ,吲哚乙酸含量高。
关键词:草酸青霉菌;PGPR;小麦;溶磷细菌
中图分类号:S476　　　　　　文献标识码:　A
97第 3 期 Aili.Tureke , et al:Growth Promotion of Wheat Seedlings by Penicillium oxalicum