Symbiotic fungi in roots of Artemisia annua with special reference to endophytic colonizers Speaker:Ting-Yin Cheng( 鄭婷尹 ) Advisor:Dr. ( 藍清隆 ) Data:2013// Z. L. YUAN, Y. C. CHEN, & X. J. MA

Introduction

Artemisia annua 1.Family : Asteraceae Genus : Artemisia 2.Morphology : fern-like leaves, bright yellow flowers, and a camphor-like scent. 3.Medicinal properties : treat fever in the ancient by Chinese; artemisinin is used for Malaria treatment. 4.Distribution : all of the world, and Asia is major.

Dark septate endophytes(DSEs) 1.Growth : the plant roots 2.Morphology : changing shapes; the common feature is the mycelium darker in color, and form a distinct diaphragm. 3. Characteristics : the plant root epidermis forming Hartig net, and mycelium intrude into Root cortex form microsclerotia or uncompleted sets of the mycelium.

DSEs Microsclerotia

Hartig net

Microsclerotia

Mycelium

Arbuscular mycorrhizal fungi 1.Phylum ： Glomeromycota （球囊菌門） 2. a type of mycorrhiza( 菌根菌 ) in which the fungus penetrates the cortical cells of the roots of a vascular plant. 3. help plants to capture nutrients such as phosphorus, sulfur, nitrogen and micronutrients from the soil.

Arbuscular mycorrhizal fungi

Malt extract agar(MEA) 1.Including 2%malt extract, 2%agar, and 50mg/l Chloromycetin. 2.Usually used to culture yeasts

PCR 1.The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a specific DNA fragment from DNA samples in vitro. 2.three steps: (1.)denaturation(92-94 ℃ ): denature dsDNA->ssDNA (2.)annealing(45-65 ℃ ): primers annealing to thr complementary ssDNAs (3.)elongation(72-74 ℃ ): DNA synthesis by DNA polymerase

Aim

Determine the occurrence and identity of symbiotic fungi in A. annua roots by microscopic and culture- based approaches with special reference to endophytic fungi, and thus provide living fungal cultures for further experimentation in vitvo.

Experimental materials

Sterile deionized water 50%(v/v) Ethanol 75%(v/v) Ethanol 5%(w/v) KOH 2% Lactic acid 0.05% (w/v) Trypan blue 50%(v/v) Glycerin 1%(w/v) Sodium hypochlorite 15% (v/v) Glycerol MEA(2% malt extract;2% agar; 50mg/l Chloromycetin) PDB Biospin Fungus Genomic DNA Extraction Kit Primers ITS1 & ITS4 PCR reactions

Experimental procedurs & Results

1.Samples collection 2.Observation & assessment 3.Isolation, purification & storage 4.Morphological & molecular identification

1.Samples collection Place ： Guangxi botanical garden of medicinal plant (E108°20’-N22°48’), China. Time : July 2008 Ways : 3 naturally regenerated and 3 planted plants growing up to 5 months and randomly collected.

2.Observation & assessment Step: Roots were cleaned by sterile deionized water and fixed in 50%(v/v) ethanol for24 hr. Roots were rinsed with distilled water three times and placed in 5% (w/v) KOH in water bath at 90 ℃ for 2 hr. Roots were rinsed with distilled water three times and kept in 2% lactic acid for 2 min.

Quickly stained in 0.05%(w/v) trypan blue in lactic acid at 50 ℃ for 5hr. De-stained in 50%(v/v) glycerin for 24hr. Longitudinal sections of roots were squashed on slide and then immersed in 50%(v/v) glycerin for microscopic analysis.

Result 1 Using a LM to investigate the colonization pattern of symbiotic fungi associated with the naturally regenerated and planted A. annua roots at 2 sampling site.

A:Vesicles were mainly distributed along root cortex. D;E:AMF extraradical hyphae. F:AMF Intradical hyphae, vesicles, and collapsed arbuscules. AMF colonization in roots of naturally regenerated A. annua Bar=20  m

DSE & other endophytic fungi colonization in roots of naturally regenerated A. annua C;E;F;G:Different structure of microsclerotia.

Mycorrhizal fungi, DSEs & other endophytic fungi inhabitating in roots of planted A. annua A:Vesicles were distributed along root cortex. D:Infection and growth of septa, melanised hyphae. G;H:Microsclerotia formed in root cortex by dark septate endophytes.

Analysis : (1.)F AM % : arbuscular mycorrhizal frequency; a ratio between root fragments colonized by AMF & the total number of root fragments examinde. (2.)F DSE % : dark septate arbuscular mycorrhizal frequence; in the case of endophytic fungi colonization. (3.)F EN % : other endophytic fungi frequency; in the case of endophytic fungi colonization, blue stain.

Result1: a low colonization frequency of arbuscular mycorrhizal fungi was detected in planted roots. Result2: a more colonization frequency of DSEs and other fungal endophytes was detected in planted roots.

3.Isolation, purification & storage Step : Sample roots were rinsed in running water. Soaked in 75%(v/v)ethanol for 40 sec. Immersed in 1%(w/v)sodium hypochlorite for 5 min.

Rinsed three times in sterile distilled water. Roots were cut into slices of 0.5~0.8cm length; and transferred to malt extract agar(MEA) plate. Incubated at 25 ℃ in permanent darkness for 4 days. Cuted off the fungal hyphae and sub-cultured for purification. By covering a culture on PDA slants with sterile liquid paraffin at 25 ℃ & by preservation in aqueous 15%(v/v) Glycerol.

4.Morphological & molecular identification By PCR Primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) Step : Fungal isolates were cultured in PDB for 4 days at 180rpm/min in a shaker incubator at 25 ℃. Mycelium were collected and ground in liquid nitrogen.

Fungal genomic DNA was extravted using Kit. Used primers ITS1 & ITS4 for amplification of the fulngal rDNA internal transcribed spacer(ITS) regions 1 & 2. Fungal genomic DNA was extravted using Kit.

Reference 1. 維基百科 - 球囊菌門 /3/ WIKI-Arbuscular mycorrhiza /3/ 叢枝菌根真菌 ent/MICR9/Micr93.htm-2013/3/31 ent/MICR9/Micr93.htm 5. WIKI-PCR 6. 百度百科 -PCRhttp://baike.baidu.com/view/2764.htm-2013/3/31http://baike.baidu.com/view/2764.htm 7.

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