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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2020, Vol. 46 Issue (4): 417-427    DOI: 10.3785/j.issn.1008-9209.2019.10.241
园艺学     
蓝莓根毛缺失的机制及内生菌根真菌的促生作用
尤式备(),徐佳慧,郭怡文,廖芳蕾,杨莉,陈文荣(),郭卫东
浙江师范大学化学与生命科学学院，浙江 金华 321004
Mechanism of root hair deficiency and growth-promoting effect of endophytic mycorrhizal fungi in blueberry
Shibei YOU(),Jiahui XU,Yiwen GUO,Fanglei LIAO,Li YANG,Wenrong CHEN(),Weidong GUO
College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
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摘要：

为探究蓝莓根毛缺失机制，以‘布里吉塔’蓝莓根系为材料，对6个根毛发生关键基因进行分离鉴定；同时，为筛选蓝莓促生内生菌根真菌并研究其促生机制，从地栽‘夏普蓝’和‘奥尼尔’蓝莓根系中分离并纯化了12株蓝莓内生菌根真菌，并回接到‘海岸’蓝莓苗进行接种试验。结果表明：根毛发生负调控基因GL2、WER、TTG均有表达，而正调控基因TRY、CPC、ETC1未见表达，这可能是蓝莓根毛缺失的主要原因。分离得到的菌根真菌主要来自于镰刀菌属（Fusarium）、踝节菌属（Talaromyces）、赤霉属（Gibberella）、青霉属（Penicillium）和曲霉属（Aspergillus），其中：A1、A4、X2及X22真菌接种显著促进了蓝莓株高增长；同时，A1、A4和X22接种显著提高了蓝莓的根系活力；A4、X2和X22接种对根际的酸化效果显著。对根际栽培基质及叶片中磷素含量分析表明，A4、X22处理组叶片总磷含量显著提高，而根际栽培基质中速效磷含量并未见降低，推断A4、X22形成的菌根可促进磷元素有效化。此外，在X22分泌物中检测到维生素B2的存在，而维生素B2与植物生长及抗逆性有关，暗示维生素B2的分泌或是杜鹃花类菌根的另一促生机制。
关键词： 蓝莓;  根毛缺失;  内生菌根真菌;  促生作用;  速效磷    
Abstract:

In order to elucidate the mechanism of root hair deficiency in blueberry, six key genes involved in root hair development were isolated and identified using ‘Brigitta’ blueberry seedlings as materials; meanwhile, to screen probiotic endophytic mycorrhizal fungi and study their mechanism of promoting seedling growth, 12 strains of endophytic mycorrhizal fungi were isolated and purified from the roots of mature blueberry bushes of ‘Sharpblue’ and ‘O’Neal’. Then these isolated fungi were separately inoculated on ‘Gulfcoast’ blueberry seedlings. The results showed that the negative regulators in root hair development, GL2, WER and TTG were expressed in root hairs, while the positive regulators TRY, CPC and ETC1 were not expressed, which may be the main mechanism resulted in root hair deficiency in blueberry. The isolated mycorrhizal fungi mainly belonged to the genera of Fusarium, Talaromyces, Gibberella, Penicillium and Aspergillus. Inoculation experiments showed the strains A1, A4, X2 and X22 significantly promoted the growth of blueberry; meanwhile, A1, A4 and X22 significantly increased root activity; A4, X2 and X22 significantly acidified the rhizospheric soil. In the A4 and X22 treatment groups, the total phosphorus contents of leaves increased significantly, but no obvious decrease was observed in the available phosphorus content in rhizosphere. Therefore, it can be inferred that the mycorrhiza formed by the strains of A4 and X22 can enhance the phosphorus availability. Vitamin B2, related to plant growth and resistance, was detected in the secretion of X22, which suggests that biosynthesis of vitamin B2 may be another plant growth-promoting mechanism of ericoid mycorrhizae.
Key words: blueberry    root hair deficiency    endophytic mycorrhizal fungi    growth-promoting effect    available phosphorus
收稿日期: 2019-10-24 出版日期: 2020-09-11
CLC:  S 663.9  
基金资助: 浙江省公益技术研究项目(2017C32046);浙江省重大科技专项计划(2016C02052-9)
通讯作者: 陈文荣     E-mail: 516646726@qq.com;cwr@zjnu.cn
作者简介: 尤式备（https://orcid.org/0000-0002-4167-201X），E-mail：516646726@qq.com
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尤式备
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引用本文:

尤式备,徐佳慧,郭怡文,廖芳蕾,杨莉,陈文荣,郭卫东. 蓝莓根毛缺失的机制及内生菌根真菌的促生作用[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(4): 417-427.

Shibei YOU,Jiahui XU,Yiwen GUO,Fanglei LIAO,Li YANG,Wenrong CHEN,Weidong GUO. Mechanism of root hair deficiency and growth-promoting effect of endophytic mycorrhizal fungi in blueberry. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(4): 417-427.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.10.241    或    http://www.zjujournals.com/agr/CN/Y2020/V46/I4/417

基因

Gene

引物序列（5′→3′）

Primer sequence (5′→3′)

退火温度

Annealing temperature/℃

CPCF: ATGGCTGACTTAGATCGCTCCT66.1
R: TTAATAAGCGTTGAGATCGGG61.0
WERF: ATGGAAGGAGAGAAGGGATACAA63.8
R: TTACAAGCTATGCCAAACAAAATC61.6
TTGF: ATGGAGAATTCAACCCAAGAATC62.0
R: TCAGACTTTTAGAAGCTGCATTTTG63.3
ETC1F: ATGGCTGACTCAGAACACTCTTC65.2
R: TCATTCGCTCGTTGAGTATCTG63.1
GL2F: ATGGCTACAGCTGGGGAC64.4
R: TCACCCGTCTTCGCATTG63.0
TRYF: ATGGACAAACGTCCCAGG62.5
R: CTAGCTTCTTTTATTGTGTTTCAT60.1
表1  本研究中使用的引物
图1  蓝莓根毛发生相关基因扩增（A）及cDNA克隆（B）电泳图
图2  蓝莓内生菌根真菌的形态学特征
图3  基于NJ法的蓝莓内生菌根真菌的系统聚类分析
图4  蓝莓内生菌根真菌回接对蓝莓株高增长的影响CK：未接种。与对照组（CK）相比，*表示在P＜0.05水平差异有统计学意义，**表示在P＜0.01水平差异有高度统计学意义。
图5  根系内生菌根真菌侵染形态A.菌丝团；B.菌丝圈；C.细胞间隙的菌丝；D.由菌丝组成的网状结构（如红色箭头所示）。

接种菌

Inoculated fungi

根系活力

Root activity/(μg/(g?h))

根际pH

Rhizosphere pH

侵染率

Colonization rate/%

CK201.94±38.85c5.32±0.03a10.33±3.06b
A1388.77±98.15a5.40±0.06a50.00±10.82a
A4290.64±77.14b5.16±0.05b45.00±4.36a
X2210.36±24.58c5.22±0.07b60.67±1.53a
X22404.54±51.50a5.18±0.01b58.67±13.80a
表2  接种不同有益内生菌对根系活力及根际pH的影响
图6  接种促生内生菌根真菌对根际速效养分及叶片总氮磷含量的影响CK：未接种。叶片总磷和总氮含量按干质量计。与对照组（CK）相比，*表示在P＜0.05水平差异有统计学意义，**表示在P＜0.01水平差异有高度统计学意义。
图7  促生内生菌根真菌培养液（A）及菌丝体抽提液（B）中核黄素的荧光定性分析
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