全 文 ：Ｒesearch Paper 研究报告
微生物学报 Acta Microbiologica Sinica
55(1) :33 － 39;4 January 2015
ISSN 0001 － 6209;CN 11 － 1995 /Q
http:/ / journals． im． ac． cn /actamicrocn
doi:10. 13343 / j． cnki． wsxb． 20140158
Screening of endophytic fungi with anti-phytopathogen
activities from Heptacodium miconioides
Lijun Wang1，Tingting Chen1，Yuanyuan Zou1，Manli Zhu1，Wenhua Yu1，Yinglao
Zhang1，2*
1 College of Chemistry and Life Science，Zhejiang Normal University，Jinhua 321004，Zhejiang Province，China
2 State Key Laboratory of Pharmaceutical Biotechnology，Nanjing University，Nanjing 210093，Jiangsu Province，China
Abstract: ［Objective］ To find anti-phytopathogen compounds from endophytic fungi associated with the endangered
species Heptacodium miconioides． ［Methods］Fungi from H． miconioides with antifungal activities were isolated according
to the plate growth inhibition method． The fungus with preferable antifungal activities was identified by morphological
identification and 5. 8S rＲNA sequence analysis． The bioactive metabolites were isolated and purified by chromatographic
methods;the structures were determined by spectroscopic analysis． ［Ｒesults］ Alternaria solani QZH 10 showed better
antifungal activity against Ｒhizoctorzia solani and Valsa mali with the inhibition rates of 89. 1% and 67. 9%，respectively．
The ethyl acetate crude extract of QZH 10 had strong antifungal activity against Magnaporthe oryzae with the rate of
100. 0% under the concentration of 100 μg /mL． Two antifungal metabolites altersolanol A and 6-O-methylalaternin were
isolated and determined from QZH 10． Altersolanol A possessed strong activity against M． oryzae with the inhibition rate of
more than 85%，6-O-methylalaternin had the mightily activity against V． mali with the inhibition rate of 100. 0% under
the concentration of 100 μg /mL． ［Conclusion］Altersolanol A and 6-O-methylalaternin are potential fungicides originated
from microorganisms．
Keywords:Heptacodium miconioides，Alternaria solani，endophytic fungi，antifungal activity，natural product
CLC number:Q935 Article ID:0001-6209(2015)01-0033-07
Supported by the National Natural Science Foundation of China (NSFC) (21002092，21272215)and by the Open Project of State Key Laboratory
of Pharmaceutical Biotechnology in Nanjing University (KF-GN-201411)
* Corresponding author． Tel:+ 86-579-82286419;E-mail:ylzhang@ zjnu． cn
Ｒeceived:28 March 2014 /Ｒevised:4 September 2014
Phytopathogen has always been a big problem in
agriculture，weakening the quality of products and
affecting the crop yield． Currently，the main measure
to resist the phytopathogen is chemical pesticides．
However， long-term use of chemical pesticides has
brought some problems， such as phytopathogen
resistance， food security， and environmental
pollution［1］． Therefore， the development of new
fungicides is imminent． Microbial control of
phytopathogen has many potential advantages，and it is
an important research direction of the new fungicides．
Endophytes can incubate in their host plants for a
Wijun Wang et al． /Acta Microbiologica Sinica(2015)55(1)
prolonged period or display disease symptoms when
their hosts are growing under adverse environmental
conditions［2］，which play a major role in providing
hosts resistance against different biotic and abiotic
stresses［3 － 4］． Ｒecent studies have demonstrated that
the endophytic fungi communities living within plant
tissues produce a wide range of metabolites with
different bioactivities that may be used as scaffolds for
the development of new drugs［5 － 7］．
Heptacodium miconioides is a rare and endangered
plant of China， which has a high economic and
scientific study value． The endangered plant H．
miconioides has some antifungal activities，it seemed
possible that its tissues might be colonized by
endophytic fungi which can produce antifungal
compounds［8］． The objectives of the present studies
were to separate the endophytic fungi from H．
miconioides， screen the antifungal activities against
several phytopathogen and isolate the active
compounds，which expect to lay a foundation to find
new natural antifungal metabolites for the development
of microbial fungicides．
1 Materials and methods
1. 1 Isolation of endophytic fungi
The fresh，healthy stems，leaves，bark samples of
Heptacodium miconioides were collected and processed
within 24 h of collection． They were washed in running
water to remove soil particles． The leaves were
sterilized by sequential immersion in 75% ethanol for 1
min，followed by 3% NaClO for 1 min and then rinsed
5 times in sterilized distilled water for 5 s each． The
stems and bark were in 75% ethanol for 3 min，3%
NaClO for 5 min followed by rinsing 5 times in
sterilized distilled water，and then dried on the sterile
filter paper． The sterilized tissues were cut into 5 cm
pieces and deposited on a Petri dish containing MEA
medium (20 g of malt，20 g of sucrose，1 g of peptone
and 20 g of agar in 1 L of distilled water)． The
material was incubated at 28℃ for 3 － 4 days and
hyphal tips of the fungi protruding from the inner
segments on the plates were further purified and
transferred to slants．
1. 2 Microbial fermentation
The fresh mycelium grown on MEA medium at
28℃ was inoculated into 250 mL Erlenmeyer flasks
containing 100 mL of ME liquid medium (20 g of
malt，20 g of sucrose，1 g of peptone in 1 L of distilled
water)，followed by shaking (200 r /min)continuously
for 7 days at 28℃ ． The broth culture was filtered to
separate the culture broth and mycelium． The culture
broth was refiltered with a 0. 45 μm bacterial filter to
obtain the sterile fermentation broth， subsequently，
which was kept at 4℃ ．
1. 3 Efficacy of fermentation broth of endophytic
fungi against phytopathogen in vitro
The fermentation broth bioactivities against
Ｒhizoctorzia solani and Valsa mali were tested
according to the plate growth inhibition method［9］． 1. 0
mL fermentation broth of each endophytic fungus was
mixed with 9. 0 mL MEA medium (50 － 60℃)to the
concentration of 10 fold dilution， 10. 0 mL MEA
medium was used as the blank control． After
inoculating the 5 mm diameter phytopathogen mycelial
disks onto the solid medium，the dishes were incubated
in the dark at 28℃ for 3 － 4 days． The formula for
counting the percentage of growth inhibition (GI)was
shown as follows:GI = {(A-B)/A － 5}× 100%，
where A was the diameter of the growth zone in the
control dish (mm) ，B was the diameter of the growth
zone in the experimental dish (mm)［10］．
1. 4 Antifungal activities of different polar solvent
crude extracts of QZH 10
To discover the antifungal activities of different
polar solvent crude extracts of the strain QZH 10，the
fermentation broth was extracted three times with
petroleum ether，ethyl acetate，1- butanol in turns at
room temperature， the remaining one was aqueous
phase． Then， the different solvent was dried by a
rotary evaporator to yield crude extract，respectively．
The antifungal activities of the different crude extracts
against Ｒ． solani and V． mali were tested by the
method described above．
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1. 5 Antifungal spectrum of the ethyl acetate
crude extract of QZH 10
The antifungal spectrum of the ethyl acetate crude
extract was initially tested against six kinds of fungi，
including Fusarium graminearum， Dothiorella
gregaria，Alternaria solani，Magnaporthe oryzae，F．
oxysporum f． sp． momordicae，F． oxysporum f． sp．
cucumerinum．
1. 6 Identification of QZH 10
QZH 10 was identified by comparing the
morphological character and 5. 8S rＲNA sequence to
those of standard record． The genomic DNA was
extracted according to the procedures of Power kit，
DNA isolation kit， and ITS1 (5-TCCGTAGGTG
AACCTGCGG-3)-ITS4 (5-TCCTCCGCTTATTGATA
TGC-3)primers set was chosen to amplify a major
part of the 5. 8S rＲNA，resulting the intense PCＲ
product of approximately 500 － 750 bp in size．
Polymerase chain reactions were carried out by using
50 μL reaction volumes which containing approximately
1. 0 μL of template DNA，39. 0 μL ddH2O，4. 0 μL
10 × PCＲ Buffer，4. 0 μL dNTPs，1. 0 μL of each
primer， 0. 2 μL Taq DNA polymerase． Cycling
parameters were:(1)94℃ for 2 min; (2)35 cycles
of 94℃ for 1 min，55℃ for 1 min and 72℃ for 2 min;
(3)72℃ for 5 min． A BLAST search was used to
search for the closest matched sequences in the
GenBank database［11］． The sequences obtained were
compared with nucleotide sequences deposited in
GenBank．
1. 7 Isolation and identification of active
compounds
The QZH 10 fermentation broth (30 L) was
extracted with ethyl acetate (5 × 30 L) at room
temperature． Evaporation of menstruum in vacuo gave a
tan oily residue， which was subjected to
chromatography over a silica-gel column eluting with
CH2Cl2 /MeOH mixtures of a stably growing polarity to
give active compounds． Structural identifications of the
bioactive metabolites were based on the spectroscopic
analyses． The electrospray ionization mass spectrometry
(ESI-MS)spectra were collected on a Mariner Mass
5304 instrument． The 1H and 13C nuclear magnetic
resonance (NMＲ)data were acquired on a Bruker
AVANCE-400 (Bruker Switzerland) spectrometer at
400 MHz［12］．
1. 8 Antifungal spectrums of the active
compounds
The antifungal spectrums of active compounds
were tested against the fungi mentioned above except
the D． gregaria． Cycloheximide was used as the
positive control．
2 Ｒesults
2. 1 Fermentation broth of endophytic fungi
against phytopathogen in vitro
Antifungal activities of all the endophytic fungi
isolated from H． Miconioides against Ｒ． solani and V．
Mali were tested by the plate growth inhibition method
(Table 1)． The endophytic fungi QZH 10，QZH 12，
QZH 20 and QZHⅡ 06 displayed potent antifungal
activities against the two phytopathogen with the
inhibition rate of more than 55% under the
concentration of 10 fold dilution of the fermentation
broth． Especially，the QZH 10 had strong inhibition
active against Ｒ． solani with the inhibition rate of
89. 1% (Figure 1)． However， the other strains
presented the low inhibition rate against the tested
phytopathogen．
2. 2 Antifungal activities of different polar solvent
crude extracts of QZH 10
As demonstrated in Table 2，the petroleum ether
and ethyl acetate phase exhibited considerable
activities against Ｒ． solani with the inhibition rate of
more than 60%，and the ethyl acetate phase showed
96. 2% radial growth inhibition rate against V． mali
under the concentration of 100 μg /mL (Figure 2)．
Therefore， the main antifungal ingredients of
fermentation broth may contain in the ethyl acetate
fraction．
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Wijun Wang et al． /Acta Microbiologica Sinica(2015)55(1)
Table 1． Inhibition rate of 10 fold dilution of the fermentation broth from the endophytic fungi of
H． miconioides against Ｒ． solani and V． mali
Strains No．
Ｒ． solani V． mali
Colony
diameter
Inhibition
rate /%
Colony
diameter
Inhibition
rate /%
Strains No．
Ｒ． solani V． mali
Colony
diameter
Inhibition
rate /%
Colony
diameter
Inhibition
rate /%
QZH 01 26. 5 ± 3. 2 25. 2 ± 8. 3 17. 4 ± 2. 4 51. 7 ± 8. 7 QZH 26 36. 9 ± 1. 4 0. 0 ± 0. 0 15. 5 ± 3. 8 58. 9 ± 9. 4
QZH 02 25. 0 ± 1. 7 30. 2 ± 4. 0 28. 1 ± 1. 3 9. 6 ± 4. 5 QZHⅡ01 27. 0 ± 0. 9 23. 3 ± 2. 8 39. 7 ± 3. 1 0. 0 ± 0. 0
QZH 03 23. 3 ± 2. 4 36. 2 ± 5. 5 19. 2 ± 0. 8 44. 5 ± 2. 9 QZHⅡ03 25. 2 ± 2. 6 29. 7 ± 8. 0 30. 9 ± 2. 7 0. 0 ± 0. 0
QZH 04 23. 8 ± 2. 7 34. 5 ± 6. 4 17. 0 ± 1. 4 53. 0 ± 4. 9 QZHⅡ04 27. 8 ± 9. 9 20. 6 ± 3. 1 17. 5 ± 2. 5 51. 0 ± 6. 0
QZH 05 19. 6 ± 1. 2 49. 1 ± 2. 8 20. 2 ± 2. 1 40. 8 ± 7. 6 QZHⅡ05 20. 3 ± 1. 8 46. 7 ± 5. 4 20. 0 ± 1. 4 41. 5 ± 4. 2
QZH 06 30. 2 ± 0. 9 12. 0 ± 4. 7 30. 2 ± 0. 8 1. 4 ± 4. 6 QZHⅡ06 9. 6 ± 0. 3 84. 1 ± 1. 0 16. 0 ± 1. 6 57. 0 ± 5. 7
QZH 07 37. 0 ± 2. 7 0. 0 ± 0. 0 29. 3 ± 3. 9 5. 0 ± 9. 5 QZHⅡ07 22. 8 ± 1. 2 38. 0 ± 3. 6 20. 2 ± 0. 6 40. 5 ± 1. 9
QZH 08 35. 6 ± 0. 9 0. 0 ± 0. 0 22. 2 ± 1. 6 32. 7 ± 5. 8 QZHⅡ08 26. 7 ± 2. 0 24. 3 ± 6. 0 40. 8 ± 2. 3 0. 0 ± 0. 0
QZH 09 36. 3 ± 1. 7 0. 0 ± 0. 0 15. 9 ± 2. 0 57. 6 ± 5. 9 QZHⅡ09 26. 0 ± 2. 3 26. 8 ± 7. 0 23. 4 ± 2. 0 28. 1 ± 7. 2
QZH 10 8. 1 ± 2. 6 89. 1 ± 6. 0 13. 2 ± 1. 9 67. 9 ± 6. 7 QZHⅡ10 24. 0 ± 1. 1 33. 9 ± 3. 4 36. 9 ± 3. 1 0. 0 ± 0. 0
QZH 11 30. 0 ± 2. 8 12. 8 ± 6. 4 30. 4 ± 1. 2 0. 7 ± 4. 2 QZHⅡ11 27. 3 ± 1. 7 22. 2 ± 5. 3 18. 8 ± 2. 5 45. 9 ± 7. 4
QZH 12 8. 7 ± 1. 0 87. 0 ± 2. 4 12. 3 ± 0. 9 71. 5 ± 3. 1 QZHⅡ12 26. 4 ± 0. 9 25. 4 ± 2. 8 15. 2 ± 2. 1 60. 3 ± 6. 1
QZH 13 8. 1 ± 1. 8 89. 1 ± 4. 3 24. 7 ± 2. 7 22. 9 ± 9. 7 QZHⅡ13 27. 1 ± 1. 6 23. 1 ± 4. 9 13. 9 ± 1. 9 65. 3 ± 5. 7
QZH 14 22. 4 ± 3. 5 39. 5 ± 8. 1 18. 5 ± 2. 6 47. 4 ± 9. 1 QZHⅡ14 27. 7 ± 1. 5 21. 0 ± 4. 7 30. 1 ± 1. 4 1. 8 ± 4. 2
QZH 15 31. 3 ± 3. 9 8. 3 ± 9. 1 22. 2 ± 1. 5 32. 9 ± 5. 2 QZHⅡ15 22. 6 ± 2. 1 38. 8 ± 6. 5 23. 0 ± 3. 2 29. 6 ± 9. 5
QZH 16 7. 1 ± 0. 5 92. 8 ± 1. 1 19. 3 ± 2. 3 44. 1 ± 8. 3 QZHⅡ16 15. 4 ± 0. 8 63. 8 ± 2. 3 28. 1 ± 2. 0 9. 7 ± 6. 0
QZH 17 23. 5 ± 2. 4 35. 5 ± 7. 6 31. 5 ± 4. 0 0. 0 ± 0. 0 QZHⅡ17 27. 9 ± 1. 9 20. 3 ± 5. 9 34. 2 ± 1. 5 0. 0 ± 0. 0
QZH 18 21. 5 ± 2. 0 42. 3 ± 6. 2 6. 7 ± 0. 6 93. 3 ± 1. 5 QZHⅡ18 11. 4 ± 1. 1 77. 6 ± 3. 4 22. 6 ± 1. 0 31. 2 ± 3. 7
QZH 19 36. 0 ± 1. 8 0. 0 ± 0. 0 7. 0 ± 1. 1 92. 2 ± 2. 7 QZHⅡ19 24. 4 ± 2. 6 32. 6 ± 7. 9 26. 3 ± 2. 4 16. 7 ± 8. 4
QZH 20 17. 5 ± 1. 9 56. 5 ± 6. 1 9. 7 ± 1. 3 81. 8 ± 3. 2 QZHⅡ21 13. 1 ± 0. 6 71. 8 ± 1. 9 36. 9 ± 1. 3 0. 0 ± 0. 0
QZH 21 34. 3 ± 2. 2 0. 0 ± 0. 0 19. 2 ± 2. 4 44. 5 ± 5. 9 QZHⅡ22 28. 4 ± 2. 6 18. 5 ± 8. 1 26. 8 ± 1. 2 15. 0 ± 4. 4
QZH 22 20. 5 ± 2. 3 46. 0 ± 5. 0 30. 2 ± 1. 6 1. 4 ± 9. 8 QZHⅡ23 19. 9 ± 3. 6 48. 0 ± 8. 0 9. 0 ± 0. 9 75. 6 ± 5. 6
QZH 23 35. 7 ± 3. 3 0. 0 ± 0. 0 12. 1 ± 3. 0 72. 3 ± 7. 3 QZHⅡ24 11. 9 ± 1. 1 76. 1 ± 3. 2 27. 0 ± 2. 8 14. 0 ± 9. 8
QZH 24 30. 3 ± 2. 4 11. 9 ± 7. 4 17. 7 ± 3. 4 50. 3 ± 8. 4 QZHⅡ25 19. 3 ± 2. 0 50. 1 ± 6. 2 24. 3 ± 2. 7 24. 6 ± 6. 7
QZH 25 39. 1 ± 1. 8 0. 0 ± 0. 0 23. 7 ± 3. 0 27. 0 ± 7. 5 CK 33. 7 ± 2. 0 30. 6 ± 2. 5
Unit of colony diameter:mm． All the values were mean values ± standard deviation of three determinations．
Figure 1． Inhibition rate of 10 fold dilution of QZH 10 fermentation
broth against Ｒ． solani．
Table 2． Inhibition rate of antifungal activities of different
polar solvent crude extracts of QZH 10 against
Ｒ． solani and V． mali (%)
Solvent phase Ｒ． solani V． mali
Petroleum ether phase 61. 8 ± 1. 1 39. 4 ± 8. 9
Ethyl acetate phase 60. 0 ± 2. 3 96. 2 ± 2. 8
Butanol phase 12. 1 ± 5. 2 49. 4 ± 9. 3
Aqueous phase 5. 0 ± 5. 6 46. 9 ± 2. 3
All the values were mean values ± standard deviation of three
determinations． The concentration of crude extract was 100 μg /mL．
Figure 2． Antifungal activity of the ethyl acetate phase of QZH 10
against V． mali．
2. 3 Antifungal spectrum test of QZH 10 ethyl
acetate crude extract
The antifungal spectrum of QZH 10 ethyl acetate
crude extract against 6 phytopathogen was performed
under the concentration of 100 μg /mL (Table 3)． The
crude extract showed strong inhibition activity against M．
oryzae with the inhibition rate of 100. 0%． However，the
crude extract presented weak activities against other tested
phytopathogen with the inhibition rate of less than 31%．
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Table 3． Inhibition activity of antifungal activities of the ethyl
acetate crude extract of QZH 10 against 6 phytopathogen
Phytopathogen
Colony
diameter /mm
Control
diameter /mm
Inhibition
rate /%
F． graminearum 28. 0 ± 1. 5 32. 8 ± 1. 6 17. 3 ± 5. 6
D． gregaria 12. 8 ± 0. 5 12. 9 ± 0. 9 0. 8 ± 6. 3
A． solani 14. 0 ± 0. 6 17. 9 ± 1. 0 30. 6 ± 4. 7
M． oryzae 5. 0 ± 0. 0 20. 8 ± 1. 0 100. 0 ± 0. 0
F． oxysporum
f． sp． momordicae
23. 4 ± 0. 4 25. 3 ± 1. 5 9. 5 ± 1. 9
F． oxysporum
f． sp． cucumerinum
22. 7 ± 1. 5 22. 1 ± 0. 8 0. 0 ± 0. 0
All the values were mean values ± standard deviation of three
determinations． The concentration of crude extract was 100 μg /mL．
2. 4 Identification of QZH 10
QZH 10 was identified based on microscopical
examination and 5. 8S rＲNA sequence analysis． The
colonies of strain QZH 10 were dry and produced
yellow pigment on the MEA agar medium． The aerial
mycelia were ourishing and combined with the
medium closely． The sporal chains were dark and the
spores were dark， elongated and separated． The
phylogenetic tree (Figure 3) indicated that the title
strain was closely related to Alternaria solani
(JF491194)，with the 5. 8S rＲNA gene sequence
similarity of 98% ． Therefore，we treated this strain as
Alternaria solani．
Figure 3． Phylogenetic tree based on the 5. 8S rＲNA sequences of the fungus QZH 10 from H． miconioides．
2. 5 Isolation and identification of the active
compounds of QZH 10
Bioassay-guided separation crude extract of QZH
10 (115. 83 g)afforded two bioactive compounds 1
(10 mg) and 2 (11 mg)． Both compounds were
characterized by spectroscopic analyses and comparison
with the literatures［13-14］． Both compounds were
identified as altersolanol A (1 ) and 6-O-
methylalaternin (2) based on the following data
(Figure 4) ，respectively．
Altersolanol A (1) :ESI-HＲMS (m/z ［2M +
Na］+ 695. 1588， calculated for C32 H32 O16 Na
695. 1588)． 1H-NMＲ (Acetone-d6)δ:1. 47 (3H，
s) ，3. 79 (1H，s) ，3. 92 (1H，d，J = 5. 9 Hz) ，
4. 00 (2H，s) ，4. 19 (1H，s) ，4. 26 (1H，s) ，4. 59
(1H，d，J = 5. 0 Hz) ，4. 77 (1H，s) ，4. 88 (1H，
d，J = 5. 0 Hz) ，6. 77 (1H，s) ，7. 12 (1H，s)． 13C-
NMＲ (Acetone-d6) δ:18. 8，21. 8，55. 9，69. 1，
70. 0，100. 0，105. 6，107. 4，109. 7，133. 7，142. 7，
164. 5，166. 4，166. 8，183. 7，189. 9．
Figure 4． Molecular structures of 1 and 2．
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Wijun Wang et al． /Acta Microbiologica Sinica(2015)55(1)
6-O-methylalaternin (2) :ESI-HＲMS (m / z［M-
H］－ 299. 0556，calculated for C16 H12 O6 299. 0556)．
1H-NMＲ (DMSO-d6)δ:2. 25 (3H，s) ，3. 91 (3H，
s) ，6. 79 (1H，s) ，7. 11 (1H，s) ，7. 50 (1H，s) ，
10. 37 (1H，s) ，12. 11 (1H，s)．
2. 6 Antifungal spectrums of 1 and 2
Under the concentration of 100 μg /mL，
compound 1 had the potent antifungal activity against
M． oryzae with the inhibition rate of more than 85%
(Table 4) ，and compound 2 had the strong antifungal
activity against V． mali with the inhibition rate of
100. 0% (Figure 5) ，which was comparable to that of
the positive cycloheximide．
Table 4． Inhibition rate of antifungal activities of monomer compounds to phytopathogen (%)
Phytopathogen
1 2 Cycloheximidea
50 μg /mL 100 μg /mL 50 μg /mL 100 μg /mL 50 μg /mL 100 μg /mL
Ｒ． solani 42. 4 ± 3. 8 60. 9 ± 3. 2 18. 5 ± 2. 4 39. 7 ± 3. 3 100. 0 ± 0. 0 100. 0 ± 0. 0
F． graminearum 46. 1 ± 5. 9 74. 5 ± 3. 8 32. 0 ± 3. 5 40. 3 ± 6. 5 91. 5 ± 1. 6 100. 0 ± 0. 0
A． solani 4. 1 ± 4. 2 0. 0 ± 0. 0 0. 0 ± 0. 0 1. 9 ± 6. 5 79. 4 ± 9. 6 89. 7 ± 8. 2
V． mali 8. 5 ± 3. 0 39. 7 ± 3. 1 3. 0 ± 1. 4 100. 0 ± 0. 0 100. 0 ± 0. 0 100. 0 ± 0. 0
M． oryzae N /Tb 86. 8 ± 3. 7 55. 3 ± 2. 9 N /T 90. 4 ± 7. 7 100. 0 ± 0. 0
F． oxysporum f． sp． cucumerinum 30. 2 ± 2. 9 37. 2 ± 6. 4 9. 3 ± 8. 9 28. 7 ± 3. 8 0. 0 ± 0. 0 12. 4 ± 6. 8
F． oxysporum f． sp． momordicae 21. 9 ± 9. 7 34. 4 ± 7. 4 0. 0 ± 0. 0 0. 0 ± 0. 0 70. 3 ± 3. 9 76. 1 ± 1. 5
aCycloheximide was co-assayed as a positive control． bN /T:not tested． All the values were mean values ± standard deviation of three determinations．
Figure 5． Antifungal activity of compound 2 against V． mali under
the concentration of 100 μg / mL．
3 Discussion and Conclusion
In summary，we performed the anti-phytopathogen
screening of endophytic fungi associated with the
endangered species H． miconioides and identified two
bioactive compounds from the fungus A． solani QZH 10．
Altersolanol A possessed strong activity against M．
oryzae， and 6-O-methylalaternin had the potent
antifungal activity against V． mali． These results
suggested that altersolanol A and 6-O-methylalaternin
had potential to be used as agricultural fungicides．
Further studies will be carried out to better understand
the mechanism of action associated with antifungal
effects． In addition，the discovery of our study provided
additional evidence that the endophytes related with
endangered plant species，may inspire the discovery of
useful metabolites with interesting bioactivities．
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基金项目:国家自然科学基金项目(21002092，21272215) ;南京大学医药生物技术国家重点实验室开放课题(KF-GN-201411)
* 通信作者。Tel:+ 86-579-82286419;E-mail:ylzhang@ zjnu． cn
作者简介:王丽君(1993 －)，女，浙江人，本科生，主要研究天然药物化学。E-mail:wlj930708@ sina． cn
收稿日期:2014-03-28;修回日期:2014-09-04
七子花内生真菌的抗菌活性筛选及其活性先导化合物的发现
王丽君1，陈婷婷1，邹园园1，朱蔓莉1，余文华1，张应烙1，2*
1浙江师范大学化学与生命科学学院，浙江 金华 321004
2南京大学医药生物技术国家重点实验室，江苏 南京 210093
摘要:【目的】从濒危植物七子花中分离出具有抗植物致病菌活性的内生真菌，并在其代谢产物中寻找具有
抗菌活性的先导化合物。【方法】采用生长速率法测定内生真菌对植物病原菌的抑制活性。通过形态学和
5. 8S rＲNA序列法对具有较好抗菌活性的内生真菌进行鉴定。运用多种色谱方法对发酵产物进行分离、纯
化，利用质谱和核磁共振谱分析鉴定出化合物的结构。【结果】茄交链孢 QZH 10 对水稻纹枯病菌和苹果树
腐烂病菌的抑制活性较好，抑制率分别为 89. 1%和 67. 9%。在供试浓度为 100 μg /mL时，QZH 10 乙酸乙酯
粗提物对稻瘟病菌具有强烈的抑制效果，抑制率为 100. 0%。从 QZH 10 中分离到 2 个单体化合物并分别被
鉴定为 altersolanol A 和 6-O-methylalaternin。在供试浓度为 100 μg /mL 时，altersolanol A 对稻瘟病菌具有较
强的抑制效果，抑制率大于 85%;6-O-methylalaternin 对苹果树腐烂病菌具有强烈的抑制效果，抑制率为
100. 0%。【结论】当前结果表明 Altersolanol A和 6-O-methylalaternin具有开发成微生物源杀菌剂的潜力。
关键词:七子花，茄交链孢，内生真菌，抗菌活性，天然产物
中图分类号:Q935 文章编号:0001-6209(2015)01-0039-07
(本文责编:张晓丽)
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