库尔勒香梨采后萼端黑斑病发病果实果肉细胞超微结构和生理活性的变化

赵晓梅，吴玉鹏，陈维维，叶 凯

（1.新疆农业科学院生物质能源研究所，乌鲁木齐 830091；2.新疆农业职业技术学院，新疆昌吉 831100）

库尔勒香梨采后萼端黑斑病发病果实果肉细胞超微结构和生理活性的变化

赵晓梅1，吴玉鹏2，陈维维1，叶 凯1

（1.新疆农业科学院生物质能源研究所，乌鲁木齐 830091；2.新疆农业职业技术学院，新疆昌吉 831100）

【目的】研究库尔勒香梨果肉细胞超微结构的差异以及生理活性的高低对萼端黑斑病发生的影响。【方法】以库尔勒香梨脱萼果和宿萼果为试材，在健康脱萼果和宿萼果果实上反接库尔勒香梨萼端黑斑病病原菌，研究接种病原菌后病斑的发病情况与果肉细胞超微结构、果实POD、SOD、PPO活性等生理活性变化的相关性。【结果】细胞超微结构和病斑直径、面积随时间的变化都证实脱萼果的发病程度较轻。脱萼果和宿萼果中POD、SOD、PPO和相对电导率的变化不同，与两种果实中病斑的发病情况不一致。【结论】同时侵染时，脱萼果病斑出现的时间较晚，受害程度较轻；脱萼果果实的PPO活性和相对电导率变化较大，而宿萼果果实的POD活性一直保持较高水平；脱萼果和宿萼果果实的SOD活性变化一样。

库尔勒香梨；萼端黑斑病；细胞超微结构；生理活性

0 Introduction

【Research significance】Korla fragrant pear is famous for strong fruit smell，skin thin and flesh crisp，sweet and refreshing，rich nutrition and other characteristics，what become high quality and unique fruit in xinjiang.But in recent years there are due to varieties of degradation and improper cultivation management measures，the number of fruit without calyx is decrease and the number of fruit with calyx is increase；According to the survey，incidence rates of fruit with calyx in korla fragrant pear postharvest calyx end black spot fruit is as high as 80%［1］，which make fruit appearance and quality gradually decline.Therefore，this paper discusses the differences of two fruit impacting on calyx end black spot，which has important practical significance.【Previous research progress】In recent years， research in calyx end black spot of korla fragrant pear has mainly focused on the disease causes，pathogenesis regularity and controltechnology［2－5］.【Entry point】Research in the differences of fruit pulp cell ultrastructure and physical activity in fruit without and with calyx has rarely been reported.【The key problems intended to be solved】This test would inoculate pathogenic bacteria extracted to healthy fruit without and with calyx.To study the onset of disease spot after inoculation，and discuss the correlation of disease happening and fruit pulp cell ultrastructure，POD，SOD and PPO activity changing.We would lay a theoretical basis to improve the quality of the fruit and commercial.

1 Materials and methods

1．1 Materials

Materials was collected from Wang De－fu 15year－old korla fragrant pear fruit trees in State Fruit Tree Resource Garden of Luntai in Xinjiang Academy of Agricultural Science.We picked eight mature pears at 7 o＇clock in the morning，Packed well back to the lab，and choosed fruit without and with calyx to test.

1．2 Handling and sampling

Korla fragrant pear postharvest calyx end black spot pathogen was extracted to inoculate respectively fruit without and with calyx the size，color，fruity and degree consistent.Starting from 1 d，it was cut width and thickness of 1.0～1.5 mm，length of 5 mm peel and pulp tissue in the middle of the same parts and fixed.It was used 3% glutaraldehyde（prepared with phosphate buffer 0.2 mol／L）to fix 12 hours at room temperature.After flushing it 1.5 h by buffer，it was used 1%osmic acid（with 0.2 mol MPBS buffer preparation of L－1）to fix 1.5 h.

Buffer was rinsed 0.5 h，alcohol was used for dehydration.Using acetone transition，and using Epon 812 epoxy resin infiltration and embedding，cutting slice thickness for 60 nm with a LEICA ULTRACUT R type ultra－thin slicing machine.Dyeing with uranyl acetate and lead citrate，using Hitachi h－600 transmission electron microscope and camera.Putting the fruit after inoculation in plastic turnover box，stored at room temperature（25℃ and 70%～80%RH）away from light environment，after 12 h，determinating initial value.

1．3 Method for determination

Disease spot diameter and area；Peroxidase（POD）and superoxide disproportionation enzyme（SOD）（reference Cao Jian－kang method［6］）. Polyphenol oxidase（PPO）activity（reference Lian Yi，Hu Jian－feng method adapted［7－11］）.Relative electric conductivity（L1／L0x 100，L1－extraction liquid conductivity after soaking；L0－conductivity of extract when kill after tissue）.

1．4 Data Processing

Application of Excel 2010 software to produce curve.

2 Results and analysis

2．1 The changes of pulp cells ultrastructure

2.1.1 Pulp cell ultrastructure of fruit without and with calyx in the early inoculation

Fig．1a Ultra－structure of flesh cell in fruit without calyx at first day after inoculation（×1 000）

Fig．1b Ultra－structure of flesh cell in fruit with calyx at first day after inoculation（×1 000）

As shown in figure 1 a，group a was pulp cell ultrastructure of korla fragrant pear fruit without calyx on 1 d inoculation.Fruit skin cell structure remained intact，oval cells for rules.There was clear line between each cell.The cell wall and cell membrane structure were clear.The outer epidermis cells arranged closely.It contained mitochondria and chloroplasts and other cell contents.Epidermal cells and pulp cells of fruit surface to the fruit in the center had clearly structured.Organelles packed closely near the cell membrane.As shown in figure 1 b，group b was pulp cell ultrastructure of korla fragrant pear fruit with calyx on 1 d inoculation.Cell structure was complete near the surface of fruit，which ap-peared a triangle，oval and other irregular shape. Fruit pulp cells presented rules oval near the stone. Cell wall，cell membrane and organelles and other structure were clear，other organelles were distributed close to the membrane.

2.1.2 Pulp cell ultrastructure of fruit without and with calyx in the middle inoculation

Fig．2a Ultra－structure of flesh cell in fruit without calyx at fourth day after inoculation（×10 000）

Fig．2b Ultra－structure of flesh cell in fruit with calyx at third day after inoculation（×60 000）

As shown in figure 2 a，group a was pulp cell ultrastructure of korla fragrant pear fruit without calyx on 4 d inoculation.Calyx end black spot pathogen was clearly visible.Compared with fruit with calyx，pathogen invasion was relatively slow.At this point，the ultrastructure showed that fruit cell had began to collapse.Mitochondria and chloroplasts and other cell organelles had disintegrated.Cell wall and cell membrane had decomposited，and boundary was disappeared.As shown in figure 2 b，group b was pulp cell ultrastructure of korla fragrant pear fruit with calyx on 3 d inoculation.Calyx end black spot pathogen was clearly visible.Fruit cell had began to collapse，mitochondria and chloroplasts and other cell organelles had disintegrated.There were starch grains and addicted to osmium particles.Cell wall and cell membrane had decomposited，boundary had disappeared.

2.1.3 Pulp cell ultrastructure of fruit without and with calyx in the late inoculation

Fig．3a Ultra－structure of flesh cell in fruit without calyx at fifth day after inoculation（×40 000）

Fig．3b Ultra－structure of flesh cell in fruit with calyx at fifth day after inoculation（×40 000）

As shown in figure 3 a，group a was pulp cell ultrastructure of korla fragrant pear fruit without calyx on 5 d inoculation.Fruit cell had began to collapse，mitochondria and chloroplasts and other cell organelles had disintegrated.Middle layer substrate in the cell walls had obviously collapsed.Most of thecell space were occupied by a vacuole.Gap between cell and cell became larger.Cell wall thickness was between 0.407 ～ 0.407 nm （x 40，000）.As shown in figure 3 b，group b was pulp cell ultrastructure of korla fragrant pear fruit with calyx on 5 d inoculation.Fruit cell had began to collapse，Most of the cell organelles were invisible.Cytoplasm were pushed aside to close to the location of the cell membrane.Cell wall thickness was between 0.614～0.674 nm（x 40，000）.

2．2 The changes of disease spot diameter and area in fruits with and without calyx

Fig．4 Changes of lesion diameter in fruits with and without calyx after inoculation

Fig．5 Changes of lesion area in fruits with and without calyx after inoculation

As shown in figure 4 and figure 5，When korla fragrant pear fruit without and with calyx were inoculated calyx end black spot pathogen.Disease spot diameter and area in two kinds of fruit were rising constantly with the time.Compared with fruit with calyx，both disease spot diameter and area in fruit without calyx havd grown slowly.It mained a lesser degree in fruit without calyx.

2．3 The changes of polyphenol oxidase（PPO）activity in fruits

Polyphenol oxidase was one kind of oxidase containing copper，which was mainly exist in the plant cell plastids and small body.When the plant tissue was infected disease or in adversity，polyphenol oxidase activity was significantly increasing，and therefore it was easy to browning.

Figure 6，when two kinds of fruit were inoculated pathogen，the PPO activity was firstly showing down，then rised in the peak and declined.After inoculation 4 d，the change of PPO activity was big in fruit without calyx.It showed that fruit without calyx was easy to browning after infestation，which might be associated with the differences of the cellular structure.

Fig．6 Changes of PPO activity in fruits with and without calyx after inoculation

2．4 The changes of peroxidase（POD）activity in fruits

Peroxidase，catalase and superoxide dismutase were known collectively as the protective enzyme system.They kept free radicals at a lower level through mutual synergy to prevent harming organisms［12］.POD could promote H2O2degradation，removed harmful free radicals in cells.

Figure 7，after pathogen was inoculated in fruit without calyx，the POD activity of the fruit basically remained unchanged on 1 to 5 d，but it was rising rapidly after 5 d；POD activity of fruit with calyx increaseed gradually，it reached the peak on 4 d，and then rose again after 5 d.This might be related to the disease degree of the fruit.Because of a lesser degree and coming on slowly in the infected fruit without calyx，so the POD activity of the emergency response was not so strong in the early stage of the disease.

Fig．7 Changes of POD activity in fruit with and without calyx after inoculation

2．5 The changes of superoxide dismutase（SOD）activity in fruits

SOD could remove free radicals，and reduced damage to the cell membrane.Figure 8，SOD activity droped rapidly on 1 d after when fruit without calyx was inoculated pathogen.From 3 to 6 d，the fruit SOD activity basicly kept in 0.010 min／（g·FW）；SOD activity also droped rapidly on 1 d after when fruit with calyx was inoculated.It was slowly rising to the peak of 0.032 min／（g·FW）on 4 d，then decreased.As a whole，there had little difference between fruit without and with calyx.

Fig．8 Changes of SOD activity in fruit with and without calyx after inoculation

2．6 The changes of relative electric conductivity in fruit

Plant cell membrane played an important role in maintaining the cell microenvironment and normal metabolism.When the plant was affected by or adverse conditions such as high and low temperature，the cell membrane woule be damaged，the permeability and conductivity increased.Membrane permeability increased the degree associated with which the size of adversity stress intensity，also be related to the strength of the plant resistance［13］.

Figure 9，The cells relative conductivity droped rapidly on 1 d after when fruit without calyx was inoculated pathogen.It was rising the peak of 11.20% on 3 d，then fell，and rose again on 5 d；The cells relative conductivity was rising slowly on 1 d after when fruit with calyx was inoculated.It was rising the peak of 8.24%on 3 d，then fell，and rose again on 5 d；The relative conductivity of fruit without calyx was higher from figure 9.But the relative conductivity of fruit without calyx had a downward trend than the initial value，fruit with calyx was on the contrary. These meaned thatthe organizational structure of fruit without calyx was more advantageous to maintain the integrity of the cell membrane.

Fig．9 Changes of relative conductivity in fruit with and without calyx after inoculation

3 Discussion

3.1 Fruit peel anthocyanin and chlorophyll content in Korla fragrant pear fruit without calyx were higher than fruit with calyx，and carotenoid content was minimum［14］.Total sugar，sucrose，soluble solids，VC content and solid acid in fruit without calyx were significantly higher than fruit with calyx.Korla fragrant pear fruit cell ultrastructure and rusty spot disease research under controlled atmosphere storage had been reported［15］.

3.2 Cell ultrastructure showed that disease spot appeared time of fruit without calyx was later than fruit with calyx，which had smaller intercellular space than fruit with calyx，these explained that fruit without calyx was normal.Disease spot diameter and area of fruit without calyx grew slower than fruit with calyx，confirmed that fruit without calyx had a lesserdegree.Measurement period，the changes of PPO activity was intense in fruit without calyx than fruit with calyx.POD activity of fruit without calyx changed smoothly within the first 5 d，but had a higher value in fruit with calyx.PPO and POD activity were pulp tissue to make corresponding response to calyx end black rot pathogen infection.From the change of both we concluded that pulp tissue responsed sensitively in fruit without calyx in early stage，which had a lesser degree.And fruit without calyx might be browning in later（after 5 d）.There had little difference in SOD activity between fruit without and with calyx.The relative electric conductivity of fruit without calyx changed violently，it might be that fruit pulp cell membrane was destroyed by pathogen infection，which caused the increasing of relative electrical conductivity.

4 Conclusion

4.1 When korla fragrant pear fruit without calyx was infected by calyx end black rot pathogen，the disease spot would appear later，and intercellular space became smaller，disease spot diameter and area grew slowlier；

4.2 PPO，POD activity of fruit without calyx increased in the late when korla fragrant pear was infected by calyx end black rot pathogen，and the pulp cells relative conductivity increased.

（

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Fruit Pulp Cell Ultrastructure of Korla Fragrant Pear Postharvest Calyx End Black Rot Disease and the Change of Main Physiological Activity

ZHAO Xiao－mei1，WU Yu－peng2，CHEN Wei－wei1，YE Kai1
（1．Research Institute of Bioenergy，Xinjiang Academy of Agricultural Sciences，Urumqi 830091，China；2．Xinjiang Vocational College of Agriculture，Changji Xinjiang 831100，China）

【Objective】To study the effects of different Korla pear flesh cell ultrastructural and physiological level on the activity of the calyx end black spot.【Method】In this experiment，Korla calyx fruit and persistent calyx fruit were used as test materials to study the correlation of disease spot incidence after inoculation of pathogen and fruit pulp cell ultrastructure，POD，SOD and PPO physiological activity.【Result】Cell ultrastructure and the disease spot diameter，area changing over time confirmed that disease spot incidence of fruit without calyx had a lesser degree.The change of POD，SOD，PPO and the relative electrical conductivity were different in fruit without and with calyx.This did not match the incidence of disease spot in two kinds of fruit.【Conclusion】When there is infection at the same time，the timing of disease spot appeared in fruit without calyx was late and had a lesser victim degree.PPO activity and the relative electrical conductivity changed a lot in fruit without calyx，and POD activity had maintained a higher level in fruit with calyx.The changes of the SOD activity were the same in fruit without and with calyx.

Korla fragrant pear；calyx end black spot；cell ultrastructure；physiological activity

436.612.1

A

1001－4330（2016）09－1640－07

10.6048／j.issn.1001－4330.2016.09.010

2016－01－26

自治区自然科学基金面上项目“新疆库尔勒香梨萼端黑斑病调查及病原菌生物学特性的研究”（2015211A030）

赵晓梅（1980－），女，甘肃金昌人，助理研究员，博士，研究方向为果树学，（E－mail）zxm2003076＠163.com

（Cotresponding author）：吴玉鹏（1979－），男，湖南株洲人，硕士，讲师，研究方向为果树学，（E－mail）wyp2003067＠163.com

Fund project：