全 文 :植物保护学报 Journal of Plant Protection, 2016, 43(2): 300 - 306 DOI: 10 13802 / j. cnki. zwbhxb. 2016 02 018
基金项目:中央高校特别资助项目(2011JS080)
∗通讯作者(Author for correspondence), E⁃mail: wxyrtl@ 163. com
收稿日期: 2014 - 11 - 11
Physiological and biochemical effects of essential oils
on seed potato storage
Wang Xuegui1,3 Shen Litao2 Yao Jianhong3 Liu Fan3 Wang Xiyao3∗
(1. Biorational Pesticide Research Laboratory, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China;
2. College of Resources and Environment, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China;
3. College of Agronomy, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China)
Abstract: To determinate the physiological and biochemical effects of essential oils (EOs) extracted from
Citrus limonum, Cinnamomum cassia, Cymbopogon citratus and Allium sativum on seed potato during
storage, the sprout suppression, weight loss, respiration, α⁃amylase, peroxidase and total soluble sugars
of Zhongshu 2 seed potato were assayed post⁃treatment 45, 60, 75 and 90 d. The results showed that the
EO of A. sativum had the strongest effect on seed potato producing sprouting rates of 1 88% -10 33% ,
respiration of 1 48 - 10 55 mg CO2·g - 1·h - 1, peroxidase activities of 353 37 - 176 23 U·g - 1·min - 1
from 45 to 90 d, weight loss of 51 67 - 105 93 mg / d, and soluble sugar content of 7 07 - 12 87 μg / g
from 60 to 90 d, respectively. A. sativum showed significant difference from the control in the sprouting
rates, respirations, peroxidase activities, weight losses and soluble sugar contents. The results indicated
that the EO of A. sativum could be a safe and effective way to prolong the storage period for the further
development of seed potato sprout inhibitor.
Key words: seed potato sprout inhibitor; Allium sativum; α⁃amylase; peroxidase activity; soluble sugar
几种精油对马铃薯种薯储藏期的生理生化影响
王学贵1,3 沈丽淘2 姚建洪3 刘 帆3 王西瑶3∗
(1.四川农业大学无公害农药研究室, 成都 611130; 2.四川农业大学资源环境学院, 成都 611130;
3.四川农业大学农学院, 成都 611130)
摘要: 为明确柠檬、肉桂、香茅以及大蒜精油对马铃薯种薯储藏期间生理生化的影响,测定了精油
处理储藏 45 ~ 90 d后,对中薯 2 号抑芽、重量损失、呼吸速率、α⁃淀粉酶、过氧化物酶以及总糖含量
的影响。 结果表明,从马铃薯种薯储藏后 45 ~ 90 d 内,大蒜精油处理种薯萌发率为 1 88% ~
10 33% ,呼吸速率为 1 48 ~ 10 55 mg CO2·g - 1·h - 1,过氧化物酶活为 353 37 ~ 176 23 U·g - 1·
min - 1,而在 60 ~ 90 d内重量损失率为 51 67 ~ 105 93 mg / d,可溶性糖含量为 7 07 ~ 12 87 μg / g,
均与对照差异显著。 研究表明,大蒜精油可作为马铃薯种薯抑芽剂延长休眠。
关键词: 马铃薯种薯抑芽剂;大蒜精油;α⁃淀粉酶;过氧化物酶;可溶性糖
At present, potato has become the fourth food
crop after rice, maize and wheat ( Zangeneh et al. ,
2010). However, there is a major obstacle that the
post⁃harvest storage period of many seed potatoes were
shorter than the time from first harvest to the second
cultivation, and lead to the seed potato sprouting
(Owolabi et al. ,2013). When the seed potatoes are
transported to field from stored place, the buds are
very easily broken. Various methods for prolonging the
period of tuber dormancy, including chemicals, low
temperature cold storage and so on, have been used.
However, the low temperature cold storage needs re⁃
frigerator and electric energy, which is expensive and
inconvenient in some mountain districts. Several com⁃
mercial chemicals have been used, such as chlor⁃
propham ( isopropyl N⁃( 3⁃chlorophenyl ) carbamate,
which is used to inhibit the potato sprouting (Mahajan
et al. ,2008). However, the irreversible inhibition on
the seed potato could lead to the low sprouting in agri⁃
cultural production (Harris,1978). So development of
seed potato sprouting is an urgent task.
Due to the safety and convenience of botanical
pesticides (Ruiu et al. ,2008), the search for natural
plant⁃derived products with inhibitory activity has be⁃
come a hot area in pesticide science ( Cho et al. ,
2007;Wang et al. ,2010;2014). Several natural prod⁃
ucts have been found to inhibit potato seed tuber sprou⁃
ting and seedling elongation, and some plant⁃derived
essential oils ( EOs) such as Rosmarinus officinalis,
Satureja thymbra and Coridothymus capitatus could
possess the properties of inhibiting sprouting ( Cizko⁃
vaet al. ,2000). Gómez⁃Castillo et al. (2013) found
peppermint and coriander essential oils possessed the
strong sprout inhibitors with inhibition rates of more
from 65% to 95% respect to their control. Owolabi et
al. ( 2013 ) also reported the inhibitions of EOs of
Monodora myristica, Cymbopogon citratus, Chenopodi⁃
um ambrosioides, Lippia multiflora and Zingiber offici⁃
nale on potato sprout growth and found that the tubers
treated with L. multiflora and C. citratus had shorter
sprout at 14 d with sprout length of 4 00 mm and 4 56
mm compared to the control of 6 18 mm. At 28 d,
Z officinale showed the strongest inhibition with the
length of 5 65 mm compared to the control of
8 75 mm.
To screen the potential sprout inhibitors, inclu⁃
ding the EOs extracted from C. limonum, A. sativum,
C. cassia, and C. citratus, on seed potatoes, and bet⁃
ter understand the mechanisms, the activities of inhibi⁃
tion sprouting were assayed, and the weight loss, in⁃
crease in respiration, peroxidase activity, α⁃amylase
activity, and soluble sugar content were also investiga⁃
ted in the study.
1 Materials and Methods
1 1 Materials
Potato variety: Zhongshu 2 seed potatoes were ob⁃
tained from the seed potato field, which were harvested
about a week and stored at the conditions of (75 ±
10)% humidity and 14 ± 2℃ .
Chemicals: Ba(OH) 2, K2H2PO4, Na2HPO4,
NaH2PO4, oxalic acid, guaiac⁃based phenolic, cit⁃
rate, phenolphthalein, Tris, EDTA, Triton X⁃100,
and DTT ( analytical reagent) were purchased from
Best⁃reagent Biochemistry Co. Ltd, Chengdu, China.
Instruments: 2000Uv⁃vis spectrophotometer, Uni⁃
co (Shanghai) Instrument Co. , Ltd. ; YP⁃1860A re⁃
frigerated cabinet, Haier Group Co. , Ltd.
1 2 Methods
1 2 1 EO extraction
Leaves of C. limonum, A. sativum, C. cassia,
and C. citratus were collected during October, 2013
from Leshan City in China. EOs were extracted from
leaves subjected to 3 - 4 h hydrodistillation using a
modified Clevenger apparatus, dried by anhydrous so⁃
dium sulfate, and stored at 4℃(Wang et al. , 2014).
1 2 2 EOs treatment
The EOs of C. limonum, C. cassia, C. citratus
and A. sativum were accurately weighted into a mass of
absorbent cotton and put into a plastic closure pocket
(10 cm × 15 cm) to achieve a concentration of 0 2
mg / mL, and without EO as a control. Four replica⁃
tions were conducted for each treatment and 100 seed
potatoes were prepared for each replication, in which
of 50 were used for the morphological indexes, inclu⁃
ding the bud numbers and weight loss in one closure
pocket. The other 50 were for the detection of physio⁃
logical and biochemical indexes, including α⁃amylase,
peroxidase and total soluble sugars in another closure
pocket. All treatments were kept in a refrigerated cabi⁃
net at 4℃ in whole darkness. When all indexes were
tested, a new mass of absorbent cotton containing 0 2
1032 期 Wang Xuegui, et al. : Physiological and biochemical effects of essential oils on seed potato storage
mg / mL EOs were put into the pockets instead of the
used absorbent cotton until the next storage point. The
indexes were measured at 45, 60, 75, and 90 d after
storage time, respectively. The sprouting rate (%) and
weight loss of per day were calculated as the following
formulas: sprouting rate (%) = (A1 - A2) / A1; weight
loss of per day (mg / d) = (W1 - W2) / T, where A1,
A2 are the numbers of total buds and sprouted buds,
respectively; W1, W2 and T are the total weight, re⁃
sidual weight and storage time, respectively.
1 2 3 Respiration and peroxidase activity assay
Seed potato respiration treated with EOs was de⁃
tected as Calegario et al. (2003) method with some
modification at 45, 60, 75and 90 d after treatment.
Five potatoes from 50 were randomly picked out, and
0 05 mol / L Ba(OH) 2 solution 50 mL was used to ab⁃
sorb CO2 transpired by potatoes into a modified dryer
made of white glass, which was sealed with a glass
cover and vaseline as quickly as possible. A titration
setup was prepared with a burette filled with 0 0227
mol / L oxalic acid solution for titrating the excess
Ba(OH) 2 at regular intervals with 1 - 2 drops of phe⁃
nolphthalein as a pH indicator, and the control was
tested with same method. Four replications were per⁃
formed for each treatment. Seed potato respirations
were determined according to the difference of volumes
of oxalic acid between the control and treatments. The
samples were processed for 30 min with a very slight
shaking motion to keep exposing a new surface of
Ba(OH) 2 to the CO2 for a completely reaction.
The peroxidase activity was measured with the
guaiac⁃based phenolic method (Xiong,2003). Three
potatoes from 50 were randomly picked out and homog⁃
enized in a mortar. Homogenate was accurately weight
1 0 g and then added 20 mmol / L K2H2PO4 5 mL fol⁃
lowed by centrifugation at 1 500 g for 10 min. The res⁃
idue was extracted with 20 mmol / L K2H2PO4 5 mL and
centrifuged again, and supernatants were combined
and transferred into a 25 mL volumetric flask, metered
with 20 mmol / L K2H2PO4 solution to scale lineand
stored at 4℃ as enzyme liquid. The liquid reaction
mixture was prepared as followed: 100 mL phosphate
Buffer ( pH 6 0 ) ( containing 0 2 mol / L Na2HPO4
12 3 mL and 0 2 mol / L NaH2PO4 87 7 mL) and 28
μL pure guaiac⁃based phenolic were added to a 50 mL
beaker and heated on a magnetic stir plate. The solu⁃
tion was cooled and 19 μL 30% H2O2 was added.
1 mL enzyme extract was added into the reaction mix⁃
ture to determine the peroxidase activity; 20 mmol / L
K2H2PO4 1 mL was added to the reaction mixture as a
control. Absorbance values were recorded at 470 nm at
one minute intervals for 30 min. A peroxidase activity
unit was as the 0 01 change of A470 per minute and it
was calculated as followed. Peroxidase activity
(U·g - 1·min - 1) = (A2 - A1) / ( T2 - T1 ) × D × 100,
where A2 - A1, T2 - T1 and D are the changes of absor⁃
bance values, times and dilution times, respectively.
1 2 4 α⁃amylase and soluble sugars assay
Soluble α⁃amylase activity was assayed by measur⁃
ing the reduction rate of sugars generated from soluble
starch (Kato⁃Naguchi & Macías,2005). Three pota⁃
toes from 50 treated with EOs were randomly selected,
and 1 0 g peeled potato was extracted using 25 mL cit⁃
rate Buffer (0 1 mol / L, pH 5 6) for 20 min, and the
homogenate was then centrifuged at 1 500 g for 10
min. The supernatant was decanted and diluted with
citrate buffer solution into a 100 mL volumetric flask,
then 10 mL of solution was aliquotted into another 50
mL volumetric flask and diluted with citrate buffer solu⁃
tion for enzymatic assay. β⁃amylase in the enzyme so⁃
lution was inactivated at 70℃ water bath for 15 min
and then cooled to 40℃ using 1% starch solution be⁃
fore measuring α⁃amylase activity. Absorbance values
at 540 nm were recorded with an 2000 Ultraviolet ana⁃
lyzer. The experiment was replicated three times.
Anthracene ketone colorimetric method ( Wen,
2003) was used to assay the soluble sugars. Soluble
sugars were extracted with a Buffer, containing 0 5
mol / L Tris, 10 mmol / L EDTA, 1% Triton X⁃100 (v /
v), 5 mmol / L DTT and 0 25% protease inhibitor
cocktail (m / m). The homogenates were centrifuged at
12 000 g for 5 min at 4℃ .
1 3 Data Analysis
All experimental data were analyzed by SPSS
17 0, and the significant differences were tested by
Duncan’ s new multiple range test (P < 0 05). The
figures of growth and development of T. molitor were
drawn by Sigma Plot 10 0.
203 植 物 保 护 学 报 43 卷
2 Results
2 1 The inhibition activities of EOs against tuber
sprouting and weight loss
The EOs of C. limonum, C. cassia, C. citratus,
and A. sativum showed very strong inhibition on tuber
sprouting, which of the A. sativum had the highest
suppression sprouting rates of 1 88% - 10 33% from
45 to 90 d, followed by C. limonum of 2 78% -
25 93% , C. cassia of 4 68% -45 11% , C. citratus
of 5 16% - 43 19% , and was significantly different
from that of the control, with sprouting rates of
28 03% -95 83% (P < 0 05, Fig. 1⁃a).
The trends of weight loss of seed potatoes treated
with EOs were similar with the sprouting rate. The
weight loss of control was the highest among the all
treatments of 59 3 mg / d, which significantly different
from other treatment (P < 0 05). The weight losses of
C. citratus, C. cassia, C. limonum and A. sativum
reached 45 2, 39 0, 28 8 and 29 6 mg / d at 45 d,
respectively, and had no significant difference with
each other (P > 0 05). Meanwhile, the weight losses
of EOs were lower than those of the control from
60 to 90 d after treatment, which of the control was
202 22 - 401 48 mg / d, compared to C. limonum of
59 4 -107 0 mg / d, C. cassia of 61 7 - 146 7 mg / d,
C. citratus of 61 7 - 184 1 mg / d and A. sativum of
51 7 -105 9 mg / d (Fig. 1⁃b).
Fig. 1 The inhibition activities of EOs against potato seed tuber sprouting and weight loss
The concentration of each treatment is 0 2 mg / mL. Data in the figure are mean ± SE.
2 2 The effects of EOs on seed potatoes respiration
and peroxidase activities
Respiration rates of seed potatoes treated with EOs
or not were all successively increased from 45 to 90 d.
The control was the highest with 6 28 - 17 38 mg
CO2·g - 1·h - 1 followed by C. citratus of 4 31 -
14 05 mg CO2·g - 1·h - 1, C. cassia of 3 94 - 14 32
mg CO2·g - 1·h - 1, C. limonum of 3 35 - 16 12 mg
CO2·g - 1·h - 1 and A. sativum of 1 48 - 10 55 mg
CO2·g - 1·h - 1 (Fig. 2⁃a). The respiration rates of seed
potatoes treated with EOs were significantly weaker
(P < 0 05) than the control during 60 to 90 d.
Compared to the control (217 69 - 57 74 U·g -1·
min -1), the peroxidase activities of seed potatoes trea⁃
ted with EOs were significantly enhanced. A. sativum
was the highest with 353 37 - 176 23 U·g - 1·min - 1
from 45 to 90 d after storage, followed by C. citratus of
295 33 - 73 73 U·g - 1·min - 1, C. cassia of 286 47 -
82 33 U·g - 1·min - 1 and C. limonum of 268 93 -
70 30 U·g - 1·min - 1 . A reduction in peroxidase activi⁃
ty correlated with the increased sprouting rate during
the 60 to 75 d storage period (Fig. 2⁃b).
2 3 Effects of EOs on seed potatoes α⁃amylase ac⁃
tivities and content of soluble sugars
The α⁃amylase activities were low at the early dor⁃
mancy stage (before 45 d), and the EOs of A. sati⁃
vum, C. cassia and C. limonum (0 06 - 0 16 mg·
g - 1·min - 1) were lower than C. citratus and the con⁃
trol (0 25 - 0 34 mg·g - 1·min - 1). Nevertheless, the
α⁃amylase activities of all treatments were greatly pro⁃
3032 期 Wang Xuegui, et al. : Physiological and biochemical effects of essential oils on seed potato storage
Fig. 2 Effects of EOs against potato seed tuber respiration and peroxidase activities
The concentration of each treatment is 0 2 mg / mL. Data in the figure are mean ± SE.
Fig. 3 Effects of EOs on seed potatoes α⁃amylase activities and content of soluble sugars
The concentration of each treatment is 0 2 mg / mL. Data in the figure are mean ± SE.
moted at the beginning of sprouting periods, especial
for C. citratus (1 23 - 1 84 mg·g - 1·min - 1) and the
control (1 77 - 1 76 mg·g - 1·min - 1). However, the
α⁃amylase activities of treatments were slightly de⁃
creased, except for A. sativum post⁃treatment 90 d
(Fig. 3⁃a).
The soluble sugar contents of tubers treated with
EOs were lower than the control at 45 d after treat⁃
ment. Although the EOs of C. citratus of 11 11 -
18 47 μg / g, C. limonum of 16 98 - 28 66 μg / g, C.
cassia of 10 33 - 20 59 μg / g and A. sativum of
7 07 - 10 78 μg / g quickly enhanced post⁃treatment
60 to 75 d, the soluble sugar contents of control were
still the highest of 22 28 - 30 88 μg / g and had signif⁃
icant difference with other treatments (P < 0 05) at 60
to 75 d. The soluble sugar contents continually in⁃
creased except for the C. limonum and control at 75 to
90 d, while the increasing quantity of soluble sugar
content of A. sativum was the lowest with 12 87 μg / g,
which of the control was 19 76 μg / g (Fig. 3⁃b).
3 Discussion
Seed sprouting is a highly complex process in
which the physiological and biochemical processes are
coordinated (Bewley,1997). According to Alexopou⁃
los et al. ( 2007 ), the weight loss of tubers after
sprouting is due to the growing sprouts having an in⁃
creased demand for carbohydrates coupled with the rel⁃
atively higher rate of water loss from sprouts. Teper⁃
Bamnolker et al. (2010) reported that mint EO could
403 植 物 保 护 学 报 43 卷
prevent potato from sprouting and non⁃treated
‘Désirée’ potato tubers lost 7 2% of their weight com⁃
pared to their treated counterparts which lost only
2 68% after 140 d of storage. Li & Bi (2012) also
found that 10 mmol / L naphthaleneacetic acid, 500
mg / kg cinnamaldehyde, 250 mg / kg acetaldehyde and
50 mg / kg ethephon could effectively control sprouting
with sprouting rates of 61% , 65% , 78% and 79% re⁃
spectively compared to the control. What’ s more, the
sprout growth of potato tuber was delayed by
10 mmol / L naphthaleneacetic acid and 50 mg / kg ethe⁃
phon, while no significant differences of weight loss ra⁃
tio were found between four kinds of new sprout sup⁃
pressants treated potato tuber and the control. The
study was consistent with the relevant reports, and the
sprouting and weight loss of seed potatoes treated with
EOs of A. sativum and C. limonum were inhibited
compared to the control.
Peroxidase is one of the important protective en⁃
zymes as superoxide dismutase and catalase, and when
the crops suffer some adverse environment, such as
dry, high slat press, anaerobic respiration and some
chemicals, the peroxidase active could increase to de⁃
fend the damage (Badawi et al. ,2004). Kwak et al.
(1996) reported that the peroxidase activity (POD) of
potato treated with ethephon increased about 20% ,
whereas paraquat slightly stimulated it soon after treat⁃
ment, but inhibited it after 13 d and which of abscisic
acid was promoted 1 78 folds compared to the control
at 18 d after treatment. Jia et al. ( 2009 ) also re⁃
searched the effects of polyethelene glycol 6000 on the
peroxidase active of potatoes, and found that the POD
activities increased at the beginning, and decreased
later. This study showed that the peroxidase activities
of all EOs treatments were stronger than the control and
consistent with the previous studies. However, a clear
reduction of peroxidase activities from 60 to 90 d was
found in the paper. The possible reason was the stimu⁃
lation of sprouting by high IAA concentrations. Be⁃
cause the peroxidase also plays a key role in modula⁃
ting seed dormancy and it could inhibit the synthesis of
indol⁃3⁃acetic acid (IAA). When the peroxidase activ⁃
ity becomes weak, the seed dormancy is broken and
the IAA level is increased and lead to sprouting (Shet⁃
ty et al. ,2002).
α⁃amylase plays a key role in degrading starch into
soluble sugars for an energy source (Babu,1994). When
subjected to adversity or stressed with toxic substances,
such as volatile Eos, seeds tended to respond with inhibi⁃
tion of α⁃amylase activity. Most of starch of seed potato
has been hydrolyzed into soluble sugars by α⁃amylase,
but the α⁃amylases activity was inhibited and the maxi⁃
mum contents of potatoes treated with EOs were delayed
compared to the control. The results were consistent with
previous reports (Babu,1994; Zhang et al. ,2002).
Some previous studies have demonstrated that
some natural aromatic hydrocarbons could inhibit
sprouting of potato tubers, which include monoterpe⁃
noids, such as D⁃carvone or ( 4S )⁃( + )⁃carvone
(Mittal et al. ,2001; Silva et al. ,2007), carvone, pi⁃
peretenone oxide, α⁃humulene ( Chauhan et al. ,
2011). Some researches have proved that the main in⁃
gredient of A. sativum was mostly diallyl disulphide,
di⁃2⁃propenyl trisulfide, and di⁃2⁃propenyl tetrasulfide
(Yang et al. ,2010; Martinez⁃Velazquezet al. ,2011;
Wang et al. ,2014). The active compounds of test EOs
in this paper is worth further investigation.
Upon completion of the 90 d storage experiment,
we also potted the seed potato treated with A. sativum
EO and found that the sprouting was not impacted but
the sprouts seemed to be much shorter and stouter than
the control. Based on the results, the EO of A. sati⁃
vum has inhibited the weight loss, respiration and
α⁃amylase activity and delayed the tube sprouting. In
general, they could be a promising way for elongating
the dormancy during potato tuber storage.
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