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Journal of Zhejiang University (Agriculture and Life Sciences)  2020, Vol. 46 Issue (4): 417-427    DOI: 10.3785/j.issn.1008-9209.2019.10.241
Horticulture     
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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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 wordsblueberry      root hair deficiency      endophytic mycorrhizal fungi      growth-promoting effect      available phosphorus     
Received: 24 October 2019      Published: 11 September 2020
CLC:  S 663.9  
Corresponding Authors: Wenrong CHEN     E-mail: 516646726@qq.com;cwr@zjnu.cn
Cite this article:

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.

URL:

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


蓝莓根毛缺失的机制及内生菌根真菌的促生作用

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GL2WERTTG均有表达,而正调控基因TRYCPCETC1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 

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
Table 1 Primers used in this study
Fig. 1 Electrophoretogram of PCR products of blueberry root hair development related genes (A) and their cDNA clones (B)
Fig. 2 Morphological characteristics of endophytic mycorrhizal fungi in blueberry
Fig. 3 Systematic cluster analysis of endophytic mycorrhizal fungi in blueberry based on NJ method
Fig. 4 Effects of inoculation of endophytic mycorrhizal fungi isolated from blueberry on increment of blueberry plant heightCK: Non-inoculation. Compared with the CK, single asterisk (*) and double asterisks (**) indicate significant and highly significant differences at the 0.05 and 0.01 probability levels, respectively.
Fig. 5 Infection morphology of root endophytic mycorrhizal fungiA. Pelotons; B. Hypha circles; C. Hyphae in intercellular space; D. Networks of hyphae (as indicated by the red arrow).

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
Table 2 Effects of inoculation of different beneficial endophytic mycorrhizal fungi on root activity and rhizosphere pH
Fig. 6 Effects of beneficial endophytic mycorrhizal fungi inoculation on available nutrients in rhizosphere and total nitrogen and total phosphorus contents of leavesCK: Non-inoculation. Total phosphorus and total nitrogen contents of leaves were calculated on the basis of the dry mass. Compared with the CK, single asterisk (*) and double asterisks (**) indicate significant and highly significant differences at the 0.05 and 0.01 probability levels, respectively.
Fig. 7 Qualitative fluorescence analysis of vitamin B2 (VB2) in culture medium (A) and mycelium extract (B) of beneficial endophytic mycorrhizal fungi
[1]   谭钺,王茂生,吕勐,等.土壤环境对蓝莓生长的影响及改善措施.山东农业科学,2015,47(3):80-84. DOI:10.14083/j.issn.1001-4942.2015.03.022
TAN Y, WANG M S, Lü M, et al. Effects of soil environment on blueberry growth and its improvement measures. Shandong Agricultural Sciences, 2015,47(3):80-84. (in Chinese with English abstract)
doi: 10.14083/j.issn.1001-4942.2015.03.022
[2]   PETERSON R L, FARQUHAR M L. Root hairs: specialized tubular cells extending root surfaces. The Botanical Review, 1996,62(1):1-40.
[3]   DOLAN L, DUCKETT C M, GRIERSON C S, et al. Clonal relationships and cell patterning in the root epidermis of Arabidopsis. Development, 1994,120(9):2465-2474.
[4]   GREBE M. The patterning of epidermal hairs in Arabidopsis: updated. Current Opinion in Plant Biology, 2012,15(1):31-37. DOI:10.1016/j.pbi.2011.10.010
doi: 10.1016/j.pbi.2011.10.010
[5]   READ D J. The biology of mycorrhiza in the Ericales. Canadian Journal of Botany, 2011,61(3):985-1004. DOI: 10.1139/b83-107
doi: 10.1139/b83-107
[6]   袁军.越橘菌根真菌分离及其对越橘生长结果的影响.重庆:西南农业大学,2005.
YUAN J. The isolation of ericoid mycorrhiza and its effects on the growth and fruit. Chongqing: Southwest Agricultural University, 2005.
[7]   BIZABANI C, DAMES J. Effects of inoculating Lachnum and Cadophora isolates on the growth of Vacciniumcorymbosum. Microbiological Research, 2015,181:68-74. DOI:10.1016/j.micres.2015.08.005
doi: 10.1016/j.micres.2015.08.005
[8]   BIZABANI C, FONTENLA S, DAMES J F, et al. Ericoid fungal inoculation of blueberry under commercial production in South Africa. Scientia Horticulturae, 2016,209(209):173-177. DOI:10.1016/j.scienta.2016.06.029
doi: 10.1016/j.scienta.2016.06.029
[9]   READ D J, STRIBLEY D P. Effect of mycorrhizal infection on nitrogen and phosphorus nutrition of ericaceous plants. Nature: New Biology, 1973,244(133):81-82.
[10]   MITCHELL D T, GIBSON B R. Ericoid mycorrhizal association: ability to adapt to a broad range of habitats. Mycologist, 2006,20(1):2-9. DOI:10.1016/j.mycol.2005.11.015
doi: 10.1016/j.mycol.2005.11.015
[11]   DOYLE J. DNA protocols for plants: CTAB total DNA isolation//HEWITT G M, JOHNSTON A. Molecular Techniques in Taxonomy. Berlin: Springer, 1991:283-293.
[12]   张彦苹,王晨,于华平,等.适于葡萄不同组织RNA提取方法的筛选.西北农业学报,2010,19(11):135-140.
ZHANG Y P, WANG C,YU H P, et al. Screening of RNA extraction methods for various grapevine organs and tissues. Acta Agriculturae Boreali-Occidentalis Sinica, 2010,19(11):135-140. (in Chinese with English abstract)
[13]   WALKER W H, FITZPATRICK S L, BARRERA-SALDA?A H A, et al. The human placental lactogen genes: structure, function, evolution and transcriptional regulation. Endocrine Reviews, 1991,12(4):316-328. DOI:10.1210/edrv-12-4-316
doi: 10.1210/edrv-12-4-316
[14]   PHILLIPS J M, HAYMAN D S. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society, 1970,55(1):158-161.
[15]   鲍士旦.土壤农化分析.北京:中国农业出版社,2005.
BAO S D. Agrochemical Analysis of Soil. Beijing: China Agriculture Press, 2005. (in Chinese)
[16]   杨小秋,高峰.纳氏比色分光光度法测定土壤中的速效氮和总氮含量.分析仪器,2014(4):29-32. DOI:10.3969/j.issn.1001-232x.2014.04.009
YANG X Q, GAO F. Determination of available nitrogen and total nitrogen in soil by Nessler’s colorimetry spectrophotometry. Analytical Instrumentation, 2014(4):29-32. (in Chinese with English abstract)
doi: 10.3969/j.issn.1001-232x.2014.04.009
[17]   邢永秀,莫遥,覃郁栋,等.凯氏定氮法与纳氏比色法测定植物组织中总氮含量的比较.教育教学论坛,2013(18):143-145.
XING Y X, MO Y, QIN Y D, et al. Comparison of Kjeldahl’s method and Nessler’s colorimetric method for the determination of total nitrogen in plant tissues. Education Teaching Forum, 2013(18):143-145. (in Chinese)
[18]   吴静萍,钱吉,郑师章.兰花菌根菌分泌物成分的初步分析.应用生态学报,2002(7):845-848.
WU J P, QIAN J, ZHENG S Z. A preliminary study on ingredient of secretion from fungi of orchid mycorrhiza. Chinese Journal of Applied Ecology, 2002(7):845-848. (in Chinese with English abstract)
[19]   LEE M M, SCHIEFELBEIN J. WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning. Cell, 1999,99(5):473-483. DOI:10.1016/s0092-8674(00)81536-6
doi: 10.1016/s0092-8674(00)81536-6
[20]   SCHELLMANN S, SCHNITTGER A, KIRIK V, et al. TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis. The EMBO Journal, 2002,21(19):5036-5046. DOI:10.1093/emboj/cdf524
doi: 10.1093/emboj/
[21]   MASUCCI J D, RERIE W G, FOREMAN D R, et al. The homeobox gene GLABRA2 is required for position-dependent cell differentiation in the root epidermis of Arabidopsis thaliana. Development, 1996,122(4):1253-1260.
[22]   KIRIK V, SIMON M, HUELSKAMP M, et al. The ENHANCER OF TRY AND CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis. Developmental Biology, 2004,268(2):506-513. DOI:10.1016/j.ydbio.2003.12.037
doi: 10.1016/j.ydbio.2003.12.037
[23]   TOMINAGA-WADA R, ISHIDA T, WADA T. Chapter two-new insights into the mechanism of development of Arabidopsis root hairs and trichomes. International Review of Cell and Molecular Biology, 2011,286:67-106. DOI:10.1016/B978-0-12-385859-7.00002-1
doi: 10.1016/B978-0-12-385859-7.00002-1
[24]   MUNITZ M S, RESNIK S L, PACIN A M, et al. Mycotoxigenic potential of fungi isolated from freshly harvested Argentinean blueberries. Mycotoxin Research, 2014,30(4):221-229. DOI:10.1007/s12550-014-0206-2
doi: 10.1007/s12550-014-0206-2
[25]   袁继鑫.野生蓝莓菌根真菌的分离、鉴定及接种效应.北京:北京林业大学,2013.
YUAN J X. The separation, identification and inoculation effect of mycorrhizal fungi isolated from wild blueberries. Beijing: Beijing Forestry University, 2013. (in Chinese with English abstract)
[26]   高丽霞,李森,莫爱琼,等.丛枝菌根真菌接种对兔眼蓝莓在华南地区生长的影响.生态环境学报,2012,21(8):1413-1417. DOI:10.3969/j.issn.1674-5906.2012.08.008
GAO L X, LI S, MO A Q, et al. Effects of inoculation of arbuscular mycorrhizal fungi on growth of rabbiteye blueberry (Vacciniumashei Reade) in South China. Ecology and Environment Sciences, 2012,21(8):1413-1417. (in Chinese with English abstract)
doi: 10.3969/j.issn.1674-5906.2012.08.008
[27]   杨雅婷,张妮娜,张飞,等.菌根真菌与接种时期对梨幼苗生长的影响.果树学报,2016,33():114-120.
YANG Y T, ZHANG N N, ZHANG F, et al. Effect of AMF strains and time of inoculating on root growth and development of Pyrus pashia seedlings. Journal of Fruit Science, 2016,33():114-120. (in Chinese with English abstract)
[28]   NEUMANN G, R?MHELD V. Chapter 14-rhizosphere chemistry in relation to plant nutrition//MARSCHNER P. Marschner’s Mineral Nutrition of Higher Plants. 3rd ed. San Diego, US: Academic Press, 2012:347-368. DOI:10.1016/B978-0-12-384905-2.00014-5
doi: 10.1016/B978-0-12-384905-2.00014-5
[29]   陈雅彬,李永强,孙琳,等.非酸性根际土壤对蓝莓铁元素吸收及其代谢相关基因表达的影响.园艺学报,2015,42(2):233-242. DOI:10.16420/j.issn.0513-353x.2014-0827
CHEN Y B, LI Y Q, SUN L, et al. Effects of non-acid rhizosphere pH on the iron elements uptakes and expressions of iron metabolism related genes in blueberry. Acta Horticulturae Sinica, 2015,42(2):233-242. (in Chinese with English abstract)
doi: 10.16420/j.issn.0513-353x.2014-0827
[30]   SPIERS J M. Effects of pH level and nitrogen source on elemental leaf content of ‘Trifblue’ rabbiteye blueberry. Journal of American Society for Horticultural Science, 1978,103(6):705-708.
[31]   LEAKE J R, READ D J. Methods in Microbiology. London: Academic Press, 1991.
[32]   吕淑芳,彭媛,江静.ACS2参与拟南芥幼苗根生长响应低磷反应的机制.植物生理学报,2017,53(8):1435-1443. DOI:10.13592/j.cnki.ppj.2017.0064
Lü S F, PENG Y, JIANG J. The involvement of ACS2 in low phosphate-mediated root growth of Arabidopsis seedling. Plant Physiology Journal, 2017,53(8):1435-1443. (in Chinese with English abstract)
doi: 10.13592/j.cnki.ppj.2017.0064
[33]   LIU Y J, HE J X, SHI G X, et al. Diverse communities of arbuscular mycorrhizal fungi inhabit sites with very high altitude in Tibet Plateau. FEMS Microbiology Ecology, 2011,78(2):355-365. DOI:10.1111/j.1574-6941.2011.01163.x
doi: 10.1111/j.1574-6941.2011.01163.x
[34]   DENG B L, DONG H S. Ectopic expression of riboflavin-binding protein gene TsRfBP paradoxically enhances both plant growth and drought tolerance in transgenic Arabidopsis thaliana. Journal of Plant Growth Regulation, 2013,32(1):170-181. DOI:10.1007/s00344-012-9285-5
doi: 10.1007/s00344-012-9285-5
[35]   NIE S J, XU H L. Riboflavin-induced disease resistance requires the mitogen-activated protein kinases 3 and 6 in Arabidopsis thaliana. PLoS One, 2016,11(4):e0153175. DOI:10.1371/journal.pone.0153175
doi: 10.1371/journal.pone.0153175
[36]   刘菲.核黄素激活烟草防卫反应和诱导对两种土传病害的抗性研究.山东,泰安:山东农业大学,2009.
LIU F. Riboflavin elicits defense responses in tobacco and induces protection against two soil-borne pathogens. Tai’an, Shandong: Shandong Agricultural University, 2009. (in Chinese with English abstract)
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