全 文 ：植物病理学报
ACTA PHYTOPATHOLOGICA SINICA摇 41(4): 385鄄392(2011)
Received date: 2010鄄10鄄30; Revised date: 2011鄄04鄄06
Foundation item: This project is financially supported by grants from the Natural Science Foundation of Sichuan Province, China
(2006B012); The Natural National Science Foundation of China (30571173); The Program for Changjiang Scholars
and Innovative Research Team in University of China ( IRT0453) .
Corresponding author: PAN Guang鄄tang, professor, major in maize genetic and breeding; E鄄mail: pangt1956@yahoo. com. cn
Biography: YUAN Guang鄄sheng (1978 - ), male, Anhui province, doctoral student, current research field in plant functional genomics.
Investigation on Histological Observation and
Protective Enzyme Activities in Ear Rot of Maize
(Zea mays L. ) after Fusarium moniliforme Infection
YUAN Guang鄄sheng, ZHAO Mao鄄jun, ZHANG Zhi鄄ming,
SHEN Ya鄄ou, PAN Guang鄄tang*
(Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Maize Research Institute,
Sichuan Agricultural University, Ya爷an 625014, China)
Abstract: To further investigate the processes involved in maize ear rot upon infection by the fungus Fusari鄄
um moniliforme (FM), the time鄄course infection was observed through scanning electron microscope (SEM)
to demonstrate pathogen progression in maize bract, then the biochemical and physiological enzyme activities
were analyzed in the two maize cultivars, Bt鄄1 and Ye478, completely resistant and significantly susceptible to
FM respectively. Microscopic observations showed that FM produced hyphae development first and then pene鄄
trated host tissues through the stoma at approximately 72 h postinoculation (hpi) . Meanwhile, the phenylala鄄
nine ammonia鄄lyases (PAL) and the reactive oxygen species (ROS) such as peroxidase (POD) were in鄄
creased promptly higher and faster in Ye478, comparing to that in resistant cultivar Bt鄄1. The content of ma鄄
londialdehyde (MDA) was higher in Bt鄄1than Ye478. Finally, the patterns of POD isozyme changed drama鄄
tically and increased three or four bands in both cultivars after infection. The results imply that the relationship
between protective enzymes activity and resistant cultivars are negatively correlated, while there was a positive
correlation between the content of MDA and resistance of the cultivars. Overall, the response to protective en鄄
zyme activities and histological observation might provide important bases to probe the resistant mechanisms,
the alteration of metabolism and the breeding research upon the infection of FM in maize.
Key Words: Zea mays; Fusarium moniliforme; ear rot; histological observation; protective enzyme
activities
串珠镰刀菌引起玉米穗粒腐病防御酶变化及其电镜观察摇 袁广胜, 赵茂俊, 张志明, 沈亚欧,
潘光堂摇 (四川农业大学玉米研究所 教育部作物基因资源与遗传改良重点实验室, 雅安 625014)
摘要: 对串珠镰刀菌(Fusarium moniliforme)侵染引起玉米穗粒腐病的防御酶活性变化和病原菌侵染过程进行研究。 采用
人工接菌的方法,分别对抗(Bt鄄1)、感(掖 478)玉米材料进行接种,取抗、感材料间隔 24 h的 6 个时间段接菌部位的苞叶组
织,分析玉米植株感病后部分防御酶、同工酶谱的动态变化,并用扫描电镜对病原菌入侵植株过程进行组织病理学观察。
扫描电镜观察发现,菌丝首先要经过 1 ~ 3 d生长后,大约在 72 h左右开始侵入气孔,并且随着时间的推移,侵入气孔的菌
丝量逐渐增多。 这说明病原菌是直接通过气孔侵入寄主苞叶组织。 同时,玉米受串珠镰刀菌侵染后,苯丙氨酸解氨酶
(PAL)和过氧化物酶(POD)的活性都是先上升后下降,在感病材料 Ye478 中 PAL 的活性要比抗病材料 Bt鄄1 中增加的更
摇
植物病理学报 41 卷
快、更高;同样对于 POD来说,在感病材料 Ye478 中的活性要比抗病材料 Bt鄄1 中的高,但变化趋势在 2 个材料中相似;而丙
二醛(MDA)的含量则相反,在感病材料 Ye478 中的活性要比抗病材料 Bt鄄1 中的低;对 POD 同工酶酶谱分析,2 个材料都
增加了 3 ~ 4 个条带,没有明显的区别,这说明玉米感病后会通过增加 POD的活性来抵御外源病菌的侵入。 总体而言 PAL
和 POD活性水平与材料抗性呈负相关;MDA与材料抗性呈正相关关系。 对玉米植株感病后防御酶活性变化的分析和病
原菌入侵寄主的电镜观察结果,可为深入研究玉米穗粒腐病抗病机制和抗病育种提供参考。
关键词: 玉米; 串珠镰刀菌; 穗粒腐; 组织病理学观察; 防御酶活性
中图分类号: S435. 131摇 摇 摇 摇 摇 文献标识码: A摇 摇 摇 摇 摇 文章编号: 0412鄄0914(2011)04鄄0385鄄08
摇 摇 Ear rot caused by Fusarium moniliforme (FM)
is a destructive disease for the decreasing yield of
maize, one of the important food crops in Asia [1,2] .
A high incidence of ear rot occurs in the moist and
warm regions of Southwest China as well as the re鄄
gion with similar longitude in other countries [3, 4] .
No doubt, improving disease resistance would be a
great advantage for farming areas where this disease
causes significant yield losses. Currently, the main
methods for controlling this disease are using bio鄄
chemical treatments and planting resistant maize cul鄄
tivars [5] . To maximize the chance on improving
disease resistance using breeding approaches, a good
understanding of the host鄄pathogen interaction is
needed, this will allow a logical selection of resistant
cultivars for breeding. In addition, the ingestion of
FM infected grain can cause severe adverse effects in
both humans and animals due to the production of
diverse and potent mycotoxins [6] .
Numerous physiological and biochemical chan鄄
ges occurred during pathogen attack, including
oxidative burst, changes in hormone levels, increa鄄
ses in soluble sugars, amino acids, organic acids and
protective proteins as well as modification of mem鄄
brane lipid composition [7鄄9] . Upon recognition of
pathogen in plant, it is usually accompanied by an
accumulation of reactive oxygen species (ROS) in
response to pathogens [10] . If the plants are not able
to control the intracellular ROS level, the membrane
lipids, proteins and nucleic acids may suffer damage
leading to death of the cells. Antioxidant enzymes
such as phenylalanine ammonia鄄lyases ( PAL ),
peroxidase ( POD ) and antioxidants have been
considered as a defensive team in plant cell, whose
combined purpose is to protect cells from oxidative
damage and maintain the levels of ROS under re鄄
stricted control [11] . Moreover, these protective en鄄
zymes have been proposed to orchestrate the estab鄄
lishment of different defensive barriers against the
pathogens. Over鄄accumulation of ROS induces rapid
death of infected areas thus blocking further spread
of the pathogens [12] . Increased PAL and POD acti鄄
vities are closely related to pathogen infection of
many plants as reported in various researches [9,13] .
These findings suggest that the induction of protec鄄
tive enzymes, such as PAL and POD are the most
common mechanisms against pathogen attack.
Recently, considerable progress has been made
in investigation of physiological and biochemical
changes involved in maize ear rots infected by FM,
including physiological and biochemical reaction re鄄
sponses, cytology of pathogen infection process,
molecular genetic analysis and isolation of disease
resistance genes leading to activation of defense
responses. In response to FM infection, the activities
of PAL and POD increased sharply at first, then
declined slowly in maize cultivar. Moreover, the
POD isozyme has increased many bands in the
research [9, 13鄄15] . Based on the histological observa鄄
tion of the pathogen infection process, macroconidia
of the pathogen might go through a period of germi鄄
nation at first, then extend and branch on host sur鄄
faces, and gradually move and infect host tissues
through the stoma at last [16] . Prior to this study,
few data is known on the changes of histological ob鄄
servation and protective enzymes with respect to FM
ear rot or possible roles in maize. The aim of this
study was to investigate the effects of increasing
PAL, POD and MDA in maize ear rot. In addition,
patterns of POD isozymes were also detected to de鄄
monstrate POD isozymes for two specific different
maize cultivars.
683
摇
摇 4 期 YUAN Guang鄄sheng,et al.:Investigation on Physiological Changes in Maize Ear Rot
1摇 Materials and Methods
1. 1 摇 Plant materials and inoculation proce鄄
dures
Both maize cultivars, resistant Bt鄄1 and suscep鄄
tible Ye478, had been identified in preliminary eva鄄
luation for many years of field work in this study.
The spores of FM were cultured on potato dextrose
agar (PDA) media for 15 d prior to collection for
inoculations. Inoculum was then prepared by wash鄄
ing conidia from the cultures and diluting a final
concentration to approximately 1. 0 伊 106 spores / mL
in water. Milky stage maize plants were inoculated
with 3 mL on each bract by injection. The inocula鄄
ted plants and mock鄄inoculated plants were grown in
the same growth chambers at Maize Research Institu鄄
te of Sichuan Agricultural University. The inocula鄄
ted and mock鄄inoculated bract tissues were collected
at 24 h intervals after inoculation for six stages.
1. 2摇 Microscopic observation
The invasion procedure of fungus on bracts was
investigated through SEM. Inoculated and mock鄄in鄄
oculated bract tissues were randomly picked by col鄄
lecting at 24 h intervals for three independent biolo鄄
gical replicates, each consisting of the independent
maize bract. Samples were initially fixed for 3 h
with 2. 5% ( v / v) glutaraldehyde in 0. 024 mol / L
phosphate buffer (pH 6. 8) . After several washes in
buffer, the specimens were post fixed with 1% (v /
v) osmium tetroxide for 5 h at 4 oC. Tissue dehy鄄
dration was carried out in a serial of ethanol dilutions
and replaced by isoamyl acetate followed by critical
point drying and carbon coating. Observations were
made on a JEM鄄100CX scanning electron micro鄄
scope operating at 25 KV.
1. 3摇 Biochemical and physiological measure鄄
ment
Total protein content in samples was determined
by Bradford method [17] . Absorbance at 595 nm of
the reaction mixture was analyzed, and protein
content was determined from a standard curve
established using known quantities of bovine serum
albumin (Sigma) . The crude extracts of PAL, POD
and MDA proteins were isolated from the inoculated
and mock鄄inoculated bract tissues using the conven鄄
tional technique with three independent biological
replicates, each consisting of the independent maize
bract [18, 19] . 1 g inoculated and mock鄄inoculated
bract tissues were triturated in 5 mL extraction buffer
(0. 2 mol / L boric acid buffer, pH 8. 8, 0. 5 mol / L
茁鄄mercaptoethanol, 2% w / v polyvinylpyrrulidone)
for PAL, 0. 05 mol / L Na2 HPO4 / NaH2 PO4 buffer
(pH 7. 0, 1% Triton X鄄100) on ice separate for
POD extraction and trichloroacetic acid ( TCA,
10% ) for MDA. Homogenates were then centri鄄
fuged at 10 000 g for PAL, MDA and 12 000 g for
POD at 4 oC for 30 min. The supernatant contained
the crude enzyme extract with PAL was analyzed by
spectrophotometer at 290 nm, and 470 nm for POD.
MDA was determined by the method of Coomassie
brilliant blue G鄄250 coloration with BSA as stand鄄
ard.
1. 4 摇 Activity detection of POD isozymes by
PAGE
POD gel activity was carried out at 72 hpi and
mock鄄inoculated controls by the Yang[20] method.
Isozymes of POD were separated on polyacrylamide
non鄄denaturating gels (10% ) at 4益 for 3 h with a
constant voltage of 150 V. After completion of elec鄄
trophoresis, gel was rinsed in water first and then in鄄
cubated in a solution composed of 0. 06% ( v / v)
H2O2, 0. 1% (w / v) benzidine and 0. 1% ( v / v)
ascorbic acid at room temperature till brown bands
appeared. The reaction was stopped by rinsing the
gels with deionized water and the gel was fixed in
acetic acid at last.
1. 5摇 Statistical analysis
All treatments were arranged in a completely
randomized collection with three replicates and the
results were then averaged. Data were assessed by
analysis of variance (ANOVA, P <0. 05) .
783
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植物病理学报 41 卷
2摇 Results
2. 1 摇 Time course of pathogen invasion be鄄
tween Bt鄄1 and Ye478
FM invasion on the maize bracts of resistant Bt鄄
1 cultivar and the susceptible Ye478 cultivar were
observed through SEM at 24 h intervals after inocu鄄
lation for six stages. During the thin and weak early
stage ( around 2 - 3 d), the hyphae of FM deve鄄
loped and expanded on the bracts surfaces of both
cultivars. After becoming significantly strong and
fine cylindrical, the hyphae gradually moved and in鄄
fected the maize bract through the stoma at approxi鄄
mately 72 hpi. Microscopic images of six time鄄
course stages are presented in Fig. 1. With the in鄄
creasing invasion of FM, more and more hyphae as鄄
sembled as mass into stoma by 120 hpi in both culti鄄
vars. However, the invasion of hyphae into stoma
was delayed in resistant cultivar Bt鄄1 as shown in
Fig. 1. This phenomenon reflected more possible
defense mechanisms in cultivar Bt鄄1 were activated
than that in Ye478.
2. 2 摇 Variation of protective enzymes activi鄄
ties in Bt鄄1 and Ye478
To study the production of enzymes involved in
the defense of FM infection, both PAL and POD
activities were detected during a 6鄄d time period. As
shown in Fig. 2鄄A, clear difference was observed in
the activities of PAL in response to the FM infec鄄
tion. The PAL activities were higher and increased
faster during the first 48 hpi in susceptible Ye478,
compared to those in resistant cultivar Bt鄄1. The
PAL activities increased promptly in the first 24 h
and peaked at 48 h in Ye478. Instead, for the Bt鄄1
cultivar, the PAL activity was measured with a slow
increase in the first 48 hpi and a peak at 72 hpi.
Meanwhile, the activities of POD increased regularly
and reached a peak at 72 hpi, and then declined
slowly in both cultivars (Fig. 2鄄B) . The curve lines
of POD activities were almost the same in both culti鄄
vars, but the activity level of POD in Ye478 was
higher than those in Bt鄄1. Hence, the activities of
defense enzymes negatively correlate with the resis鄄
tance of the cultivars.
2. 3 摇 Effects of MDA content in Bt鄄1 and
Ye478
During the FM infection at six time point, the
MDA content increased sharply in the first 48 hpi in
resistant cultivar Bt鄄1, compared to that in suscepti鄄
ble Ye478. The MDA content peaked at 48 hpi,
then slowly decreased in the following time point in
Bt鄄1. Meanwhile, the MDA content increased
slightly in the first 48 hpi and peaked at 72 hpi, then
Fig. 1摇 Electronic microscope images of the hyphea invasion procedure in stomas of both
Bt鄄1 and Ye478 cultivars for each time point ( 伊1300)
The first row is resistant cultivar Bt鄄1, and the second row is susceptible Ye478. The stomas are marked with arrows.
883
摇
摇 4 期 YUAN Guang鄄sheng,et al.:Investigation on Physiological Changes in Maize Ear Rot
Fig. 2摇 Protective enzymes change during a 6鄄d time period in both Bt鄄1 and Ye478 cultivars
A: PAL activity after inoculation; B: POD activity after inoculation. Values are average of three biological samples for each time point.
declined slowly in Ye478. As shown in Fig. 3, the
content of MDA in Bt鄄1 was higher than those in
Ye478. This implied that there was a positive corre鄄
lation between content of MDA and resistance of the
cultivar.
Fig. 3摇 Effects of MDA content for each time
point in Bt鄄1 and Ye478
2. 4 摇 Patterns of POD isozymes in Bt鄄1 and
Ye478
To further investigate the biochemical and phy鄄
siological change in different Bt鄄1 and Ye478, the
POD isozymes were analyzed at 72 hpi and mock鄄in鄄
oculated controls. On the gel, more than five or six
POD isozyme bands were visualized at 72 hpi,
whereas only two or three of them were observed in
mock controls. Since the equal amounts of enzyme
extracts were loaded on polyacrylamide gels, the
bands of POD isozyme changed dramatically and in鄄
creased three or four bands with almost the same
tendency in both cultivars ( Fig. 4 ) . The result
demonstrated that the host might be increase POD
activity and isozyme bands to resist the exogenous
pathogen attack for protecting the host organism.
Fig. 4 摇 Patterns of POD isozymes profiles at
72 hpi and mock controls in both Bt鄄1
and Ye478 cultivars
983
摇
植物病理学报 41 卷
3摇 Discussion
Maize ear rot is apathogen鄄induced disease
which affects multiple cellular activities, including
various physiological changes, membrane integrity,
DNA鄄protein interaction, and gene expression[21] .
Upon recognition of the pathogen infection in plant,
there is a defensive system in plants, that is, plants
have an internal protective enzymes catalyzed clean鄄
up system that elaborates enough to avoid injuries of
active oxygen. This system can guarantee normal
cellular function or orchestrate the establishment of
different defensive barriers against the pathogens [22] .
ROS over鄄accumulation can induce rapid cell death
of infected areas, thus blocking further spread of the
biotrophic pathogens [12] .
Numerous studies have shown that theprotective
enzymes activities were positively correlated with the
resistant cultivars [9, 13] . But some results were dif鄄
ferent from this for the protective enzymes activities
increasing higher and faster in susceptible cultivar
than those in resistant cultivar [23鄄25] . Some others
got no correlation in this relationship [26] . These
differences might result in researchers using different
experimental materials, tissues or treatment. As ob鄄
served in this study, significant increase of the PAL
and POD activities upon FM infection strongly sug鄄
gested that defensive responses were activated in the
FM鄄infected host. Furthermore, we found that en鄄
zymes activities in response to FM infection were
distinctly different in resistant maize cultivar Bt鄄1
and susceptible maize cultivar Ye478 (Fig. 2) . Ac鄄
tivities of protective enzymes in susceptible cultivar
responded stronger than those in resistant cultivar,
indicating that the relationship between protective en鄄
zymes activity and resistant cultivars was negatively
correlated. Our result was consistent to previous re鄄
ports in pathogen infection that limiting pathogen
growth in susceptible plant led to programmed cell
death (PCD) [27] . This phenomenon was consistent
with the microscopic observation that the FM inva鄄
ded tissue cells of susceptible cultivar slightly earlier
than resistant cultivar. It was likely that the pathogen
invasion of bract tissues was delayed by unknown
mechanisms in the resistant cultivar. A lower induc鄄
tion of ROS levels and a delay in invasion of FM in
the resistant cultivar might be the reason to prevent
pathogen invasion and progression. In addition, the
increased bands in the pattern of the POD isozymes
showed a similar tread compared to the quantitative
changes of POD activity assaying in both cultivars
(Fig. 4) . Although there was same pattern between
the two cultivars at 72 hpi and mock鄄inoculated con鄄
trols, further investigation on other time points is re鄄
quired to determine the actual POD isozymes pat鄄
terns in both cultivars.
It seems that althoughthe increased activities of
the protective enzymes PAL and POD might contri鄄
bute to the resistant mechanism of ear rot maize
against FM infection, the content of MDA was
closely correlated to host鄄pathogen interaction. MDA
was one of the ultimate products as a result of lipid
peroxidation damage by free radicals [19] . Due to the
lower activities of the protective enzymes in resistant
Bt鄄1 cultivar, the content of MDA increased more
significantly than those in susceptible cultivar Ye478
for the accumulated free radicals. From this point,
content of MDA in resistant cultivar responded stron鄄
ger than in susceptible cultivar, indicating positive
correlationship between content of MDA and resis鄄
tant cultivars.
In conclusion, the results clearly suggested that
in maize ear rot, under FM infection, the activities
of PAL, POD and the change of MDA content in
bract tissues basically increased at the beginning and
declined after a peak during the 6鄄d periods. More鄄
over, the patterns of POD isozyme changed signifi鄄
cantly between 72 hpi and mock controls. All these
physiological and biochemical changes, including
microscopic observation on pathogen infection
process, might indicate that defensive response
mechanisms activated in the FM鄄infected host. The
lower activity of the protective enzymes, the higher
the content of MDA in resistant cultivar Bt鄄1, which
093
摇
摇 4 期 YUAN Guang鄄sheng,et al.:Investigation on Physiological Changes in Maize Ear Rot
indicated that the relationship between protective en鄄
zymes activity and resistant cultivars was negatively
correlated, whereas positive correlationship between
content of MDA and resistant cultivars. This conclu鄄
sion was consistent with previous reports and it also
denoted a better defense mechanisms in resistant cul鄄
tivar than in susceptible cultivar. It showed that the
ability of the plant to resist pathogen was connected
with the activities of protective enzymes with their
defensive function and resistant cultivars. The FM鄄
responsive physiological changes in both cultivars
presented here are valuable resource for further
breeding studies for selection of important maize cul鄄
tivars in maize ear rot.
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