Identification and characterization of new Muscodor endophytes from gramineous plants in Xishuangbanna, China

Abstract The endophytic fungi Muscodor spp. produce volatile organic compounds (VOCs) which can inhibit and even kill pathogenic fungi, bacteria, and nematodes. Nine endophytic fungal strains, isolated from the shoots of gramineous plants including Arthraxon hispidus, Eleusine indica, Oplismenus undulatifolius, and Oryza granulata, were identified as Muscodor through phylogenetic analysis of the internal transcribed spacer. Through an SPSS K‐means cluster analysis, the nine Muscodor strains were divided into four groups based on the antifungal activities of the VOCs produced by these fungi determined by a two‐section confrontation test. The first group contains the strains Y‐L‐54, W‐S‐41, Y‐S‐35, W‐T‐27, and Y‐L‐56, which showed the strongest activity. The second and third groups contain W‐S‐35 and Y‐L‐43, which showed stronger and moderate activity, respectively. The fourth group contains W‐S‐38 and N‐L‐7, which were the weakest in inhibiting the tested pathogens. Thirty‐five compounds and the relative amounts of VOCs were determined by SPME‐GC‐MS and comparison with the NIST14 mass spectrometry database and Agilent MassHunter qualitative and quantitative analyses. These 35 compounds were classified into two different categories: (a) the product of fatty acid degradation, and (b) the intermediate and final metabolite of the metabolic pathway with the precursor of mevalonic acid. SPSS clustering analysis showed that the chemical components of VOCs might be correlated with their bioactivity rather than their phylogenetic assignment and some of the identified compounds might be responsible for antifungal activity. In conclusion, new Muscodor endophytes were recorded in tropical gramineous plants and a number of strains showed remarkable bioactive properties. Therefore, they have important potential applications in the fields of plant disease control.


| INTRODUC TI ON
Xylariaceous fungi are the dominant group of endophytic fungi. Strobel et al. found that xylariaceous fungal isolate 620 can produce volatile organic compounds (VOCs) with strong antimicrobial activity, has intertwined rope-like mycelia, and does not produce spores. Therefore, the genus Muscodor was established based on its mycelia type and its production of VOCs (Strobel, Dirkse, Sears, & Markworth, 2001;Worapong et al., 2001). Muscodor has a significant inhibitory effect or even lethal effect on a variety of pathogens (fungi, bacteria, and nematodes), therefore Muscodor has important potential applications in the fields of agriculture and environmental protection (Strobel, 2006). Muscodor albus strain QST 20799 and three end products, andante, arabesque, and glissade, were proposed for postharvest fruit, seed, and soil-borne diseases control. This was approved by the United States Environmental Protection Agency in

| Endophytic fungi isolation and storage
The gramineous plants were sampled during 2015 and 2016 from Xishuangbanna in Yunnan province of China (E 100°32′-100°44′, N 22°04′-22°17′). The samples were placed in zip-lock plastic bags, stored in a box with ice and transported to laboratory within 48 hr after sampling. Healthy plant tissues were rinsed with tap water, then immersed in 75% ethanol for 3-5 min and then in 1% sodium hypochlorite for 8-10 min, and finally rinsed thrice with sterile distilled water. The surface disinfected tissues were cut into segments of about 5 mm length and the segments were placed on MEA plate (2% malt extract agar with 50 mg/L chloramphenicol). The plates were then incubated at 25°C in darkness.
Fungal hyphae emerging from the segments were transferred to new potato dextrose agar (PDA) plates for purification. The purified cultures were grown on PDA slant, then covered with sterile liquid paraffin, and deposited in Fungal Biology Laboratory of Zhejiang University.

| Phylogenetic analysis based on the internal transcribed spacer of ribosomal DNA sequence
The internal transcribed spacer (ITS) was PCR-amplified using the universal primers, ITS-1 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′) (White, Bruns, Lee, & Taylor, 1990). The PCR amplification products were purified and sequenced bidirectionally on an ABI 3730 sequencer (Applied Bio-Systems) according to Zhang et al. (2010). The ITS sequences of the type strains of the known Muscodor species were retrieved from the National Center for Biotechnology Information's (NCBI) GenBank.
ITS sequences obtained in this study were aligned with sequences of known Muscodor species using clustal X 2.1 (Larkin et al., 2007) and manually corrected using GeneDoc (Nicholas & Nicholas, 1997).
Sequences obtained in this study were deposited in NCBI GenBank with Accession Number MG309792-MG309800.
Phylogenetic analyses were carried out using maximum parsimony (MP) approach with PAUP* v. 4.0b10 (Swofford, 2002) and Bayesian analyses (BI) approach with MrBayes v3.2.6 (Ronquist et al., 2012). jModelTest was used to compare the likelihood of different nested models of DNA substitution and select the best-fit model for the dataset. Likelihood settings from the best-fit model (TrN + G) were selected by AIC using jModeltest. The branches were indicated with MP bootstrap proportion (MPBP) and BI posterior probability (BIPP).

| Determination of the colony characteristics and optimal growth temperature
The tested Muscodor strains (Table 1) were incubated on PDA medium at 25°C for activation. The mycelial disks (5-mm diameter) were excised from the edge of the Muscodor colony, one disk was inoculated on PDA in the center of the Petri dish (9-cm diameter). The inoculated Petri dishes were kept in darkness, with three kept at each of the following temperatures: 15, 20, 23, 25, 28, 30, 35, and 40°C, respectively. After incubation for 15 days, the colony characteristics were observed and the diameters were recorded. The antifungal activity of the VOCs produced by the tested

| Determination of the components and the relative amounts of VOCs
Muscodor strains were incubated on PDA in Petri dishes with two sections as described above. The PDA plate without Muscodor was considered a control. The VOCs emitted by Muscodor were extracted with an SPME syringe (SUPELCO) 50/30 mm divinylbenzene/carboxen/polydimethylsiloxane on StableFlex fiber.
VOCs were analyzed by GC-MS (Agilent 6890N/5975B) following procedures described in Zhang et al. (2010). The data were processed using the Agilent MassHunter workstation (Agilent).

| Data statistical analysis
The growth temperature of the Muscodor endophytes pathogenic fungi were analyzed using an SPSS K-means cluster analysis. The components and the relative amounts of VOCs produced by these Muscodor strains were analyzed through SPSS clustering analysis with the nearest neighbor element and Pearson correlation, with a range from 0 to 1.

| Phylogenetic analysis of the endophytic fungal strains
Phylogenetic analysis based on ITS sequences using MP and BI The growth temperature of the tested Muscodor strains cultivated on PDA medium is shown in

| Antifungal activity of the tested Muscodor strains
The antifungal activity of the tested Muscodor strains was shown in  Among the targeted pathogens, P. ultimum was the most sensitive to VOCs produced by Muscodor. When exposed to the VOCs produced by Muscodor, P. ultimum was completely inhibited and even killed by seven of the VOCs. Penicillium digitatum (sensitive to six VOCs) and B. cinerea (sensitive to five VOCs) also showed sensitivity , which showed the strongest antifungal activity, are marked with dark blue and blue, respectively. Cluster C (strain W-S-35), which showed stronger antifungal activity, is marked with light blue. Cluster B (strain W-S-38), which showed the weakest antifungal activity, is marked with orange. Cluster E (strains N-L-7 and Y-L-43), which showed weak or the weakest antifungal activity, is marked with yellow and partially inhibited F. oxysporum and P. diospyr. The third group contains strain Y-L-43, which showed weak antifungal activity. It completely inhibited P. ultimum, moderately inhibited B. cinerea, and weakly or did not inhibit F. oxysporum, P. digitatum, and P. diospyr.
The fourth group, contains strains W-S-38 and N-L-7, which were the weakest at inhibiting the five tested pathogens.

| The components and the relative amounts of VOCs produced by the tested Muscodor strains
Thirty-five compounds and the relative amounts of VOCs were determined (Table 4), which were classified into two categories. One is the product of fatty acid degradation, such as methyl isobutyrate, 3-methyl-1-butanol, 2-methylpropanoic acid, 3-methyl-1-butyl acetate, 1-octen-3-ol, 3-octanone, and 2-nonanone. The other category is terpenes, such as monoterpe-   and Cluster E (strains N-L-7 and Y-L-43) showed weak or the weakest antifungal activity. The peak area percentage of VOC components for the five clusters was obtained (Figure 4), and the specific compounds (com1, com3, com5, com6, com8, com 9, com 10, com 12, com22, com25, and com35) in the clusters marked with varying shades of blue might be responsible for antifungal activity, and these compounds are mostly esters, alcohols, and small molecular weight acids, in particular, methyl isobutyrate and 2-methylpropanoic acid, which were produced by all highly active strains (strains W-S-41, Y-L-54, Y-S-35, W-T-27, Y-L-56, and W-S-35). The most active group of compounds identified in this study are consistent with the previous study that the esters, alcohols, and acids produced by Muscodor spp.

| D ISCUSS I ON
are remarkable bioactive properties (Mitchel et al., 2008;Strobel et al., 2001). And we would express appreciations to the Naban River Watershed National Nature Reserve for collecting samples.

CO N FLI C T O F I NTE R E S T
The authors state that there are no conflicts of interest.