生物多样性 ›› 2019, Vol. 27 ›› Issue (10): 1122-1131.doi: 10.17520/biods.2019180

• 研究报告 • 上一篇    下一篇

新疆不同来源金黄壳囊孢的多样性

赵颖1, 马荣1, 尹永香2, 张志东3, 田呈明4, *()   

  1. 1 新疆农业大学林学与园艺学院, 乌鲁木齐 830052
    2 新疆农业大学林业研究所, 乌鲁木齐 830052
    3 新疆农业科学院微生物应用研究所, 乌鲁木齐 830091
    4 北京林业大学省部共建森林培育与保护教育部重点实验室, 北京 100083
  • 收稿日期:2019-05-29 接受日期:2019-08-09 出版日期:2019-10-20
  • 通讯作者: 田呈明 E-mail:chengmt@bjfu.edu.cn
  • 基金项目:
    国家自然科学基金(31460198);中央高校基本科研业务费专项资金(2019GJZL01)

Diversity of Cytospora chrysosperma from different hosts in Xinjiang

Zhao Ying1, Ma Rong1, Yin Yongxiang2, Zhang Zhidong3, Tian Chengming4, *()   

  1. 1 College of Forestry and Horticulture, Xinjiang Agricultural University, Urumqi 830052
    2 Forestry Research Institute, Xinjiang Agricultural University, Urumqi 830052
    3 Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091
    4 Key Laboratory for Silviculture and Conservation of Ministry of Education College of Forestry, Beijing 100083
  • Received:2019-05-29 Accepted:2019-08-09 Online:2019-10-20
  • Contact: Tian Chengming E-mail:chengmt@bjfu.edu.cn

为明确新疆不同寄主及地理来源的金黄壳囊孢(Cytospora chrysosperma)的多样性, 探讨种内亲缘关系和多样性差异。作者通过记录菌株在PDA培养基上的菌落颜色、形状、子实体形态等特征, 并应用Biolog-FF技术及ISSR分子标记技术, 比较了来自新疆5个地区5种寄主上的47株金黄壳囊孢的培养特征、生理生化特征及遗传多样性。结果表明47株金黄壳囊孢依据培养特征可划分为15种类型。不同类型菌株在碳源利用及代谢能力上存在差异, 各菌株对碳源的利用数量随着培养时间的增长逐渐增多。菌株882利用的碳源数量最多, 培养120 h可利用28种不同碳源, 碳源代谢能力中等; 菌株812-1利用的碳源数量最少, 培养120 h仅利用7种碳源, 代谢能力较低; 菌株1074-2、847、934、891-1、896、740具有单独利用碳源的能力。基于遗传相似性系数进行聚类分析, 结果显示遗传相似性系数为0.58时, 47个菌株被划分为两大类群, 其中第二类群菌株的培养特征为: 菌落白色、子实体较小且分布密集。供试金黄壳囊孢的多样性主要受自身遗传结构的影响, 不同寄主种类和地理来源对多样性的影响不显著。

关键词: 金黄壳囊孢, 培养性状, Biolog-FF技术, ISSR分子标记

Cytospora chrysosperma is a very important pathogenic fungus that can be infects many different trees. Currently, the diversity, phylogenetic and genetic relationships of C. chrysosperma remain unclear as host differences could cause substantial genetic and diverse differences among C. chrysosperma strains. To analyze the genetic, phylogenetic, and current diversity of C. chrysosperma, physiological and biochemical characteristics and genetic tests were performed from different hosts in Xinjiang. Using molecular markings Biolog-FF and ISSR, 47 strains of C. chrysosperma have been divided into 15 types. Strains were cultured and displayed varying culture characteristics and carbon source utilization and metabolic capacity traits. Each strain tended to utilize more carbon over time, however, strains varied in their carbon source preferences and metabolic capacities. Using cluster analysis, 47 strains of C. chrysosperma divided into two clusters based on genetic similarity (0.58), suggesting genetic structure is essential to understanding C. chrysosperma diversity, as host species and geographical origins were not significant.

Key words: Cytospora chrysosperma, cultural characteristics, Biolog-FF, ISSR

表1

本研究中供试菌株的相关信息"

菌株 Isolate 寄主 Host 采集时间 Collecting time 采集地点 Location
319-1 苹果 Malus sp. 2011/8/7 昌吉吉木萨尔县 Jimusar County, Changji Prefecture
790-2 苹果 Malus sp. 2015/6/28 塔城地区塔城市 Tacheng, Tacheng Prefecture
908 苹果 Malus sp. 2015/7/17 塔城地区乌苏市 Wusu, Tacheng Prefecture
936 苹果 Malus sp. 2015/8/4 塔城地区塔城市 Tacheng, Tacheng Prefecture
701 榆树 Ulmus sp. 2015/4/18 吐鲁番市鄯善县 Shanshan County, Turpan City
1155 榆树 Ulmus sp. 2015/8/20 昌吉州奇台县 Qitai County, Changji Prefecture
600-2 胡杨 Populus euphratica 2012/8/20 喀什地区泽普县 Zepu County, Kashgar Prefecture
626 新疆杨 Populus alba 2012/8/20 喀什地区喀什市 Kashgar City, Kashgar Prefecture
686-1 山杨 Populus davidiana 2014/8/31 塔城地区塔城市 Tacheng City, Tacheng Prefecture
686-2 山杨 Populus davidiana 2014/9/1 塔城地区塔城市 Tacheng City, Tacheng Prefecture
702 小叶杨 Populus simonii 2015/4/18 吐鲁番市鄯善县 Shanshan County, Turpan City
737 钻天杨 Populus nigra 2015/6/4 哈密地区哈密市 Hami City, Hami Prefecture
739 钻天杨 Populus nigra 2015/6/4 哈密地区哈密市 Hami City, Hami Prefecture
740 钻天杨 Populus nigra 2015/6/4 哈密地区哈密市 Hami City, Hami Prefecture
742 钻天杨 Populus nigra 2015/6/4 哈密地区哈密市 Hami City, Hami Prefecture
793 俄罗斯杨 Populus russkii 2015/6/29 塔城地区塔城市 Tacheng City, Tacheng Prefecture
882 北京杨 Populus beijingensis 2015/7/22 塔城地区乌苏市 Wusu City, Tacheng Prefecture
891-1 俄罗斯杨 Populus russkii 2015/7/17 塔城地区乌苏市 Wusu City, Tacheng Prefecture
891-3 俄罗斯杨 Populus russkii 2015/7/17 塔城地区乌苏市 Wusu City, Tacheng Prefecture
904 大叶杨 Populus lasiocarpa 2015/7/16 塔城地区乌苏市 Wusu City, Tacheng Prefecture
914-1 杨树 Populus sp. 2015/7/21 塔城地区乌苏市 Wusu City, Tacheng Prefecture
934 新疆杨 Populus alba 2015/8/3 塔城地区乌苏市 Wusu City, Tacheng Prefecture
1074-2 胡杨 Populus euphratica 2015/8/26 吐鲁番市托克逊县 Toxon County, Turpan City
1076 胡杨 Populus euphratica 2015/8/26 吐鲁番市托克逊县 Toxon County, Turpan City
812-1 柳树 Salix sp. 2015/7/1 塔城地区塔城市 Tacheng City, Tacheng Prefecture
835-1 柳树 Salix sp. 2015/7/11 塔城地区托里县 Tori County, Tacheng Prefecture
839-2 柳树 Salix sp. 2015/7/2 塔城地区塔城市 Tacheng City, Tacheng Prefecture
839-3 柳树 Salix sp. 2015/7/2 塔城地区塔城市 Tacheng City, Tacheng Prefecture
847 柳树 Salix sp. 2015/7/6 塔城地区裕民县 Yumin County, Tacheng Prefecture
856-1 疏锯齿柳 Salix serrulatifolia 2015/7/29 塔城地区沙湾县 Shawan County, Tacheng Prefecture
755-2 红瑞木 Swida alba 2015/6/4 哈密地区哈密市 Hami City, Hami Prefecture
874-2 旱柳 Salix matsudana 2015/7/26 塔城地区沙湾县 Shawan County, Tacheng Prefecture
895-2 柳树 Salix sp. 2015/7/17 塔城地区乌苏市 Wusu City, Tacheng Prefecture
896 灰柳 Salix cinerea 2015/7/14 塔城地区托里县 Tori County, Tacheng Prefecture
897 柳树 Salix sp. 2015/7/14 塔城地区托里县 Tori County, Tacheng Prefecture
901 疏锯齿柳 Salix serrulatifolia 2015/7/14 塔城地区托里县 Tori County, Tacheng Prefecture
919-1 旱柳 Salix matsudana 2015/7/22 塔城地区塔城市 Tacheng City, Tacheng Prefecture
933 金丝垂柳 Salix × aureo-pendula 2015/8/3 塔城地区塔城市 Tacheng City, Tacheng Prefecture
935 金丝垂柳 Salix × aureo-pendula 2015/8/3 塔城地区塔城市 Tacheng City, Tacheng Prefecture
942 旱柳 Salix matsudana 2015/8/4 塔城地区塔城市 Tacheng City, Tacheng Prefecture
944-2 柳树 Salix sp. 2015/8/10 哈密地区哈密市 Hami City, Hami Prefecture
976-1 柳树 Salix sp. 2015/8/10 五家渠市 Wujiaqu City
976-2 柳树 Salix sp. 2015/8/10 五家渠市 Wujiaqu City
863 旱柳 Salix matsudana 2015/7/29 塔城地区沙湾县 Shawan County, Tacheng Prefecture
921 柳树 Salix sp. 2015/7/31 塔城地区塔城市 Tacheng City, Tacheng Prefecture
922 疏锯齿柳 Salix serrulatifolia 2015/7/31 塔城地区塔城市 Tacheng City, Tacheng Prefecture
930-1 疏锯齿柳 Salix serrulatifolia 2015/8/3 塔城地区塔城市 Tacheng City, Tacheng Prefecture

表2

本研究中ISSR引物序列"

编号
No.
引物
Primer
引物序列(5′-3′)
Primer sequence (5′-3′)
退火温度
Annealing temp. (℃)
1 IJ-5 GAGAGAGAGAGAGAGATG 52
2 IL-7 ACAACACACACACACAC 52
3 IJ-7 GGGTGGGGTGGGGTG 52
4 IJ-9 AGATGTGTGTGTGTGTG 52
5 IJ-8 ATAAGAGAGAGAGAGAG 52
6 ID-8 GTGCGTGCGTGCGTGC 52
7 IL-3 CACGAGAGAGAGAGAGA 52
8 IQ-2 CCAAACCACCACCACCACCA 53
9 IJ-3 AGAGAGAGAGAGAGAGTC 53
10 IJ-4 GAGAGAGAGAGAGAGATC 53

表3

本研究中PDA平板上金黄壳囊孢形态性状观测项目及赋值"

观察项 Item 代表值 Value
0 1 2 3 4 5
菌落颜色
Colony color
白色
White
米黄色
Light yellow
土黄色
Khaki
灰色
Gray
深灰色
Dark gray
桔红色
Orange
菌落形状
Colony shape
圆形生长
Circular growth
辐射状生长
Radial growth
- - - -
表面形态
Surface morphology
菌丝稠密
Hyphae dense
菌丝稠密
Hyphae dense
菌丝稠密
Hyphae dense
菌丝稠密
Hyphae dense
菌丝稠密
Hyphae dense
菌丝稀疏
Hyphae sparse
平伏
Flat exhibition
轻微凸起
Slight raised
轻微凸起
Slight raised
凸起
Raised
凸起
Raised
轻微凸起
Slight raised
无轮纹
No concentric ring
无轮纹
No concentric ring
有轮纹
Concentric ring
无轮纹
No concentric ring
有轮纹
Concentric ring
无轮纹
No concentric ring
有/无子实体
Sporulation
子实体小
Small sporophore
子实体小
Small sporophore
子实体大
Larger sporophore
子实体大
Larger sporophore
- -
密集型
Intensive
稀疏型
Sparse
稀疏型
Sparse
聚生型
Concentrated
- -
色素分泌
Pigment secretion
米白色
Creamy-white
米黄色
Light yellow
土黄色
Khaki
灰色
Gray
黑色
Black
桔红色
Orange

图1

47株金黄壳囊孢培养性状聚类图。A-O表示具有不同培养性状特征的分支; 数字代表菌株编号, 菌株信息见表1。"

图2

不同类型金黄壳囊孢碳源利用数量及代谢活性随时间变化图(菌株编号含义见表1)"

表4

本研究中不同类型金黄壳囊孢对主要碳源的利用情况(菌株编号含义见表1)"

碳源种类
Carbon source type
吸光度值 Absorbance value (Ci-R) 平均吸光
度值
Average absorbance value
812-1 897 1074-2 686-2 921 922 839-2 847 934 882 702 891-1 896 740 935
吐温80 Tween 80 0.735 0.715 0.296 0.525 0.334 0.472 0.332 0.305 0.349 0.290 0.598 0.458 0.401 0.812 0.700 0.488
苦杏仁苷 Amygdalin - 0.282 0.274 0.322 0.276 0.583 0.653 0.254 0.418 0.284 0.396 0.475 0.493 0.313 0.330 0.357
D-阿拉伯糖 D-Arabinose 0.685 0.632 0.821 0.521 0.266 0.709 0.624 0.250 0.419 0.272 0.499 0.557 0.425 0.426 0.307 0.494
L-阿拉伯糖 L-Arabinose 0.688 0.627 0.670 0.568 0.285 0.724 0.453 0.375 0.582 0.266 0.629 0.496 0.285 0.259 0.304 0.481
糊精 Dextrin 0.392 0.686 0.661 0.782 0.503 0.447 0.438 0.345 0.414 0.264 0.543 0.522 0.469 0.479 0.369 0.488
D-果糖 D-Fructose - 0.678 0.891 0.669 0.401 0.404 0.471 0.303 0.503 0.282 0.616 0.578 0.406 0.259 0.726 0.479
α-D-葡萄糖 α-D-glucose 0.385 0.669 0.500 0.766 0.334 0.379 0.572 0.301 0.347 0.464 0.618 0.689 0.535 0.287 0.551 0.493
D-甘露糖 D-Mannitol - 0.899 0.563 0.741 0.336 0.312 0.314 0.510 0.992 0.610 0.297 0.508 0.314 0.251 0.416 0.471
D-核糖 D-ribose 0.658 0.691 0.451 0.613 0.541 0.892 0.562 0.510 0.349 0.554 0.432 0.401 0.526 0.276 0.353 0.521
水杨苷 Salicin - 0.394 0.534 0.323 0.342 0.359 0.335 0.523 0.318 0.282 0.463 0.579 0.551 0.331 0.334 0.378
蔗糖 Sucrose - 0.28 0.430 0.255 0.349 0.498 - 0.305 0.349 - 0.356 0.252 - 0.343 0.518 0.262
D-木糖 D-xylose 0.756 0.686 0.270 0.592 0.336 0.518 0.423 0.347 0.319 0.666 0.293 0.592 0.406 0.356 0.514 0.472
富马酸 Fumaric acid - 0.354 0.361 0.378 0.436 0.381 - 1.522 0.314 0.595 0.280 0.625 - 0.294 0.517 0.404
琥珀酸 Butanedioic acid - 0.371 0.360 0.255 0.348 0.331 - 0.400 0.279 0.346 0.649 0.535 - 0.284 0.326 0.299
L-天门冬酰 L-asparagine - 1.246 0.460 0.578 0.436 0.304 0.306 0.453 0.258 0.329 0.284 0.410 0.260 0.265 0.954 0.436
平均吸光度值
Average absorbance value
0.287 0.614 0.503 0.526 0.368 0.488 0.366 0.447 0.414 0.367 0.464 0.512 0.338 0.349 0.481

表5

本研究中存在单独利用碳源的菌株及利用种类"

菌株
Strain
碳水化合物
Carbohydrate
羧酸及其衍生物
Carboxylic acids and derivatives
氨基酸
Amino acid
磷酸盐类
Phosphates
胺类
Amine
1074-2 - - L-谷氨酸
L- glutamate
L-丙胺酰胺
L-alamide
-
847 - 溴代丁二酸
Bromosuccinic acid
- - -
934 - - - - 腺苷
Adenosin
891-1 - L-乳酸
L-lactic acid
L-苯丙氨酸
L- phenylalanine
- -
896 L-海藻糖
L-trehalose
- - - -
740 - ρ-羟基苯乙酸
ρ-hydroxy acid
琥珀酰胺酸
Succinamic acid
- -
L-天门冬氨酸
L-aspartic acid

图3

47株金黄壳囊孢的ISSR聚类分析图(当遗传相似性系数为0.58时, 47个菌株被分为类群I和类群II; 菌株编号同表1)"

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[1] 徐陈贤, 刘福秀, 周晓云, 王克荣. (2004) 中国东部栗疫病菌dsRNA群体的多样性. 生物多样性, 12(3): 370-376.
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