Rhizospheric Fungi of Panax Notoginseng: Diversity and Antagonism to Host Phytopathogens
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J Ginseng Res xxx (2015) 1e8 Contents lists available at ScienceDirect Journal of Ginseng Research journal homepage: http://www.ginsengres.org Research article Rhizospheric fungi of Panax notoginseng: diversity and antagonism to host phytopathogens q q Cui-Ping Miao 1, , Qi-Li Mi 1,2, , Xin-Guo Qiao 1, You-Kun Zheng 1, You-Wei Chen 1, Li-Hua Xu 1, Hui-Lin Guan 3, Li-Xing Zhao 1,* 1 Key Laboratory of Microbial Diversity in Southwest China of Ministry of Education and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming, China 2 Technology Center, China Tobacco Yunnan Industrial Co., Ltd, Kunming, China 3 School of Energy and Environment Science, Yunnan Normal University, Kunming, China article info abstract Article history: Background: Rhizospheric fungi play an essential role in the plantesoil ecosystem, affecting plant growth Received 12 January 2015 and health. In this study, we evaluated the fungal diversity in the rhizosphere soil of 2-yr-old healthy Received in Revised form Panax notoginseng cultivated in Wenshan, China. 4 May 2015 Methods: Culture-independent Illumina MiSeq and culture-dependent techniques, combining molecular Accepted 14 June 2015 and morphological characteristics, were used to analyze the rhizospheric fungal diversity. A diffusion test Available online xxx was used to challenge the phytopathogens of P. notoginseng. Results: A total of 16,130 paired-end reads of the nuclear ribosomal internal transcribed spacer 2 were Keywords: fungal diversity generated and clustered into 860 operational taxonomic units at 97% sequence similarity. All the opera- fi Illumina MiSeq tional taxonomic units were assigned to ve phyla and 79 genera. Zygomycota (46.2%) and Ascomycota Panax notoginseng (37.8%) were the dominant taxa; Mortierella and unclassified Mortierellales accounted for a large propor- rhizosphere soil tion (44.9%) at genus level. The relative abundance of Fusarium and Phoma sequences was high, accounting for 12.9% and 5.5%, respectively. In total, 113 fungal isolates were isolated from rhizosphere soil. They were assigned to five classes, eight orders (except for an Incertae sedis), 26 genera, and 43 species based on morphological characteristics and phylogenetic analysis of the internal transcribed spacer. Fusarium was the most isolated genus with six species (24 isolates, 21.2%). The abundance of Phoma was also relatively high (8.0%). Thirteen isolates displayed antimicrobial activity against at least one test fungus. Conclusion: Our results suggest that diverse fungi including potential pathogenic ones exist in the rhizosphere soil of 2-yr-old P. notoginseng and that antagonistic isolates may be useful for biological control of pathogens. Copyright Ó 2015, The Korean Society of Ginseng, Published by Elsevier. All rights reserved. 1. Introduction most complex environments that are influenced by plant roots and active microhabitat where plant roots and microbes interact Soil microbial communities display high structural, genetic, [3,4]. Composition of the rhizosphere microbiota can negatively and functional diversity, and play extremely complex roles in or positively influence plant traits such as stress tolerance, many aspects of soil ecosystems, such as biotic interactions and health,development,andproductivity[5,6].Conversely,host nutrient cycling [1]. Microbial diversity in soil is an important plantsareabletoshapetheirownrhizospheremicrobiomeby factor determining soil health and is considered one of the main adjusting environmental factors such as pH, soil nutrients, and drivers in soil suppressiveness [2]. The rhizosphere is one of the root architecture [5], and recent studies have shown that plant * Corresponding author. Key Laboratory of Microbial Diversity in Southwest China of Ministry of Education and Laboratory for Conservation and Utilization of Bio- Resources, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, China. Tel.: þ86 8 716503 3539; fax: þ86 8 716503 3539. E-mail addresses: [email protected], [email protected] (L.-X. Zhao). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) whichq permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. These authors contributed equally to this work. 1226-8453/$ e see front matter Copyright Ó 2015, The Korean Society of Ginseng, Published by Elsevier. All rights reserved. http://dx.doi.org/10.1016/j.jgr.2015.06.004 Please cite this article in press as: Miao C-P, et al., Rhizospheric fungi of Panax notoginseng: diversity and antagonism to host phytopathogens, Journal of Ginseng Research (2015), http://dx.doi.org/10.1016/j.jgr.2015.06.004 2 J Ginseng Res 2015;-:1e8 genotype also has a great influence on the structure of rhizo- 2. Materials and methods sphere microbial communities [7e9]. Fungi and fungus-like or- ganisms form one of the most diverse groups of the Eukarya, 2.1. Site and sampling and represent an essential functional component of soil micro- bial communities [10]. Under unfavorable conditions, some Soil samples were collected in a P. notoginseng plantation (N fungi can cause plant diseases and sometimes even total loss of 23350,E103400) in Wenshan, Yunnan, in early June 2013. Soil the crop yields. In many instances, these diseases are caused by adhering around the roots was gently taken off, put into a sterile a complex of fungal species. At the same time, other fungal plastic bag, and transported to the laboratory within 24 h. Soil groups can antagonize phytopathogens, decompose plant resi- samples were preserved at À80C and 4C before use. dues, supply nutrients for plants, and stimulate plant growth [11]. 2.2. Soil DNA extraction and MiSeq sequencing The culture-dependent approach has been applied to analyze the fungal diversity for several decades [12]. In the past 20 yrs, DNA was extracted from 0.5 g frozen soil sample using the many molecular methods, such as terminal-restriction fragment PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, length polymorphism (T-RFLP), random amplified polymorphic USA), according to the manufacturer’s instructions. The nuclear DNA (RAPD), single-strand conformation polymorphism (SSCP), ribosomal internal transcribed spacer 2 (ITS2) region was amplified denaturing gradient gel electrophoresis (DGGE), and real-time according to the newly developed primers fITS9 and ITS4 [28]. quantitative PCR detecting system (Q-PCR), have widely been used Amplifications were carried out in a total volume of 50 mL using to study the diversity and composition of soil microbial commu- 10 ng of DNA, 5 mL10Â polymerase chain reaction (PCR) buffer, 5 mL nities [13,14]. With methodological advances, high-throughput deoxy-ribonucleoside triphosphate (dNTP) (10mM each), 2.5 unit sequencing techniques, such as 454 pyrosequencing and MiSeq Plantium Taq, and 0.5 mL each of Bar-PCR primer (50mM) and Primer by Illumina (San Diego, CA, USA), have been considered more R (50mM). PCR cycling conditions were as follows: 5 min at 94C; 25 powerful tools for estimating microbial communities [14,15]. cycles of 30 s at 94C, 30 s at 58C, 30 s at 72C; and 7 min at 72C. Compared to the 454 pyrosequencing, MiSeq sequencing has The PCR products were purified and run using a MiSeq Benchtop for shorter time for generating good-quality paired-end reads and a 2 Â 300 bp paired-end sequencing (Illumina). The quality check much higher throughput at a low price [15e17]. The length of reads was performed using PRINSEQ-lite 0.19.5. All sequences were can reach 2 Â 300 bp. Due to lower costs and greater throughput, aligned using UCLUST v1.1.579 (http://www.drive5.com/uclust/ Illumina MiSeq sequencing is increasingly being utilized to esti- downloads1_1_579.html), and complete linkage clustering was mate the diversity and composition of microbial communities in used to define operational taxonomic units (OTUs) with 97% iden- various ecosystems. tity as a cutoff. Rarefaction analysis was performed using MOTHUR Panax notoginseng (Burk.) F. H. Chen (Araliaceae), a well- v.1.22.2 [29]. Diversity in the sample was estimated using the known traditional Chinese medicinal plant mainly cultivated in ShannoneWeiner index and the species richness was expressed as the mountainous areas in Yunnan and Guangxi, is widely used the number of OTUs by the nonparametric indices abundance- for the treatment of cardiovascular diseases, inflammation, based coverage estimate (ACE) and Chao1 [30]. In addition, tax- trauma, and internal and external bleeding due to injury, and has onomy assignment was performed using the Ribosomal Database also been used as a tonic [18,19]. Grown under shaded conditions Project (RDP) classifier [31]. with high humidity for at least 3 yrs, cultivation of P. notoginseng is easily affected by the soil environment, and P. notoginseng is 2.3. Isolation of fungi also susceptible to a number of soil-borne diseases [20].Among the diseases, the root-rot disease complex caused by a single Fungal isolation was carried out using the dilution method. Soil pathogen or a combination of pathogens is most destructive sample (10 g) was mixed with 90 mL sterile water in a conical flask, À À because it results in yield reduction, or no harvest, and a lower and the soil solution was diluted from 10 1 to 10 3 after shaking for À content of active ingredients [21]. The reported pathogens 1 h in an oscillator. A volume of 0.1 mL soil solution (10 3)was include Alternaria panax, Alternaria tenuissima, Cylindrocarpon coated evenly on potato dextrose agar with penicillin sodium salt destructans, Cylindrocarpon didynum, Fusarium oxysporum, Fusa- (100 mg in 1 L medium) and kanamycin sulfate (50 mg in 1 L me- rium solani, Phytophthora cactorum, Phoma herbarum,and dium) to suppress bacterial growth. All plates were incubated at Rhizoctonia solani [22]. Fungal pathogens, endophytes, and room temperature for 1 wk or until fungal growth was observed.