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Introduction
Solanum rostratum Dunal. is an annual weed with a strong capacity for propagation and adaptation. A notorious invader, it also serves as the primary host of the potato leaf-roll virus and Leptinotarsa decemlineata (potato beetles), which pose substantial threats to biodiversity and the environment in China (Zhao et al. 2013, Liu et al. 2020). Additionally, S. rostratum contains abundant amounts of secondary metabolites, primarily flavonoids, alkaloids, steroids, and other compounds (Liu et al. 2020).
Endophytic fungi live in plants for all or part of their lives without harming the host (Ripa et al. 2019). They may affect a plant's ability to reproduce, grow, or resist abiotic stress or natural enemies (Rho et al. 2018). To the best of our knowledge, previous papers largely concentrated on the biological traits and phytochemical profile of S. rostratum and there is no report about the diversity of endophytic fungi of this invasive plant. The main goal of this study is to explore the community of the fungal endophytes of S. rostratum. Identification of the endophytes may help explain the invasive success of S. rostratum from the perspective of plant-microbe interaction; these endophytes are also potentially valuable resources of bioactive substances that have various biological activities.
Materials and methods
Forty-five mature S. rostratum plants at flowering stage were collected on June 28, 2018 from 3 different locations (15 plants from each location) in Urumqi and Changji city of Xinjiang province: location 1: 43º 55'60'' N, 87º 20'41'' E (Loc-1); location 2: 43º 56'0'' N, 87º 20'41'' E (Loc-2); location 3: 43º 46'14'' N, 87º 46'31'' E (Loc-3). Endophytes were isolated using a culture-dependent method within two days of collection of plants. Surface sterilization of plant parts (roots, stems, and leaves) and isolation of the endophytes were conducted following the protocol of Schulz et al. (1993). Colonization rate (CR) was counted by following Petrini et al. (1982).
DNA of the endophytic isolates was extracted by using the DNA Extraction Kit for fungi (Solarbio Life Sciences, Beijing, China), according to the manufacturer’s instructions (Abd-Elsalam et al. 2003). PCR amplification of the rDNA ITS (internal transcribe spacer) region was conducted with the use of ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') fungal primer pairs (White et al. 1990). The sequences of the fragments were identified using the basic local alignment search tool BLAST (http://www.ncbi.nlm.nih.gov/) of the NCBI and Mycobank (https://www.mycobank.org) databases. Fungal identities were generated by sequence alignment analysis with those previously submitted to GenBank.
Results and discussion
In total, 176 endophytic fungal isolates corresponding to 44 OTUs (operational taxonomic units) were isolated and were classified into 12 genera (Tab. 1). Among the isolates, 55 (31.25%) were obtained from leaves, 55 from stems (31.25%), and 66 from roots (37.50%); 34 out of 44 OTUs had between 97.14% and 100.00% sequence similarity with relevant entries in Mycobank and GenBank databases, whereas OTU1, OTU15, OTU16, OTU21, OTU23, OTU25, OTU26, OTU30, OTU37 and OTU44 had between 70.13% and 95.87% sequence similarity with species belonging to the genera Aspergillus, Penicillium, Microascus, Purpureocillium and Mucor (Tab. 1), indicating they might be potential new species. Penicillium and Aspergillus were the dominant genera of the endophytic fungal community (Cheng et al. 2018). OTU23 (closest hit Purpureocillium lilacinum CBS 284.36) was chosen for further study on its secondary metabolites due to its high plant growth regulatory activity, which resulted in the isolation and identification of 3 bioactive compounds, i.e., adenosine, cerevisterol, and thymine, which were found to possess significant plant growth regulatory activity (Kuchkarova et al. 2020).
Tab. 1. List of identified endophytic fungi isolated from Solanum rostratum plant parts. aBLASTN max score; blevel of identification for pairwise alignments by calculating using the Martinez-Needleman-Wunsch algorithm; clevel of similarity for pairwise alignments with the closest match, using the NCBI and Mycobank database; Accession number of the closest database match; daccession number of the closest database match. OUT - operational taxonomic unit.
| Best Blast hit | Number of isolates | ||||||||||||
| OTU | Accession no. | Closest taxa match | Score a | Query coverage (%) b | Ident (%) c | Accession no d | By tissue type | By location | Total observed | ||||
| Leaf | Stem | Root | 1 | 2 | 3 | ||||||||
| 1 | ON149677 | Aspergillus lentulus | 556 | 94 | 85.21 | PWQ2395 | 0 | 0 | 1 | 1 | 0 | 0 | 1 |
| 2 | ON149678 | Penicillium oxalicum | 843 | 95 | 100.00 | FMR 14261 | 6 | 4 | 18 | 18 | 4 | 6 | 28 |
| 3 | ON149679 | Pichia kudriavzevii | 711 | 96 | 99.36 | CNRMA6.98 | 1 | 0 | 0 | 1 | 0 | 0 | 1 |
| 4 | ON149680 | Aspergillus quadrilineatus | 786 | 92 | 99.40 | IHEM 22705 | 1 | 1 | 0 | 1 | 0 | 1 | 2 |
| 5 | ON149681 | Aspergillus rugulosus | 762 | 92 | 99.20 | UOA/HCPF 10020 | 2 | 0 | 3 | 5 | 0 | 0 | 5 |
| 6 | ON149682 | Emericella nidulans | 775 | 94 | 98.22 | WM 06.100 | 4 | 2 | 0 | 6 | 0 | 0 | 6 |
| 7 | ON149683 | Aspergillus creber | 637 | 94 | 97.42 | FMR 14364 | 1 | 0 | 0 | 1 | 0 | 0 | 1 |
| 8 | ON149684 | Fusarium verticillioides | 775 | 94 | 100.00 | IHEM 9835 | 0 | 1 | 0 | 1 | 0 | 0 | 1 |
| 9 | ON149708 | Penicillium citrinum | 795 | 96 | 99.60 | NRRL 1841 | 0 | 0 | 2 | 1 | 1 | 0 | 2 |
| 10 | ON149685 | Aspergillus niger | 857 | 94 | 100.00 | WM 10.76 | 7 | 0 | 1 | 7 | 0 | 1 | 8 |
| 11 | ON149686 | Aspergillus nidulans | 805 | 95 | 99.42 | WM 11.60 | 0 | 0 | 2 | 1 | 1 | 0 | 2 |
| 12 | ON149687 | Penicillium brasilianum | 843 | 94 | 100.00 | FMR 14296 | 0 | 1 | 0 | 1 | 0 | 0 | 1 |
| 13 | ON149688 | Aspergillus oryzae | 852 | 95 | 99.81 | WM 10.120 | 0 | 0 | 1 | 1 | 0 | 0 | 1 |
| 14 | ON149689 | Aspergillus tubingensis | 848 | 94 | 100.00 | IHEM 17440 | 3 | 3 | 2 | 4 | 0 | 4 | 8 |
| 15 | ON149690 | Penicillium rolfsii | 695 | 95 | 95.10 | FMR 14307 | 0 | 0 | 1 | 1 | 0 | 0 | 1 |
| 16 | OM698374 | Microascus cirrosus | 166 | 62 | 70.13 | FMR 12256 | 0 | 1 | 0 | 1 | 0 | 0 | 1 |
| 17 | ON149691 | Penicillium chrysogenum | 835 | 94 | 100.00 | FMR 14008 | 14 | 19 | 15 | 5 | 15 | 28 | 48 |
| 18 | ON149692 | Fusarium pseudonygami | 735 | 89 | 100.00 | U34563 | 0 | 0 | 1 | 1 | 0 | 0 | 1 |
| 19 | ON149693 | Fusarium oxysporum | 732 | 93 | 98.77 | UOA/HCPF AB82 | 0 | 0 | 1 | 1 | 0 | 0 | 1 |
| 20 | ON149694 | Aspergillus terreus | 848 | 92 | 100.00 | WM 03.218 | 0 | 0 | 2 | 2 | 0 | 0 | 2 |
| 21 | ON149695 | Aspergillus calidoustus | 667 | 95 | 95.87 | UOA/HCPF 9236 | 1 | 0 | 0 | 1 | 0 | 0 | 1 |
| 22 | ON149696 | Aspergillus fumigatus | 863 | 95 | 100.00 | ATCC 1022 | 1 | 0 | 0 | 1 | 0 | 0 | 1 |
| 23 | ON149697 | Purpureocillium lilacinum | 624 | 76 | 95.59 | CBS 284.36 | 4 | 5 | 2 | 4 | 3 | 4 | 11 |
| 24 | ON149698 | Penicillium coprophilum | 817 | 99 | 99.24 | FMR 13998 | 1 | 1 | 0 | 1 | 1 | 0 | 2 |
| 25 | ON149699 | Penicillium glabrum | 516 | 75 | 94.12 | FMR 14292 | 0 | 2 | 0 | 2 | 0 | 0 | 2 |
| 26 | ON149700 | Penicillium frequentans | 513 | 75 | 94.09 | FMR 14318 | 0 | 1 | 0 | 1 | 0 | 0 | 1 |
| 27 | ON149701 | Talaromyces pinophilus | 816 | 94 | 99.42 | FMR 14017 | 0 | 0 | 1 | 1 | 0 | 0 | 1 |
| 28 | ON149702 | Aspergillus aculeatus | 795 | 92 | 100.00 | CBS 172.66 | 0 | 0 | 4 | 1 | 0 | 3 | 4 |
| 29 | ON149703 | Paecilomyces lilacinus | 791 | 83 | 98.85 | WM 04.457 | 0 | 1 | 1 | 0 | 2 | 0 | 2 |
| 30 | ON149704 | Aspergillus brasiliensis | 732 | 95 | 95.48 | ATCC MY-A4553 | 1 | 0 | 0 | 0 | 1 | 0 | 1 |
| 31 | ON149705 | Aspergillus flavus | 836 | 93 | 100.00 | PWQ 2335 | 4 | 0 | 0 | 0 | 4 | 0 | 4 |
| 32 | ON149706 | Penicillium echinulatum | 770 | 94 | 98.48 | FMR 13945 | 0 | 0 | 1 | 0 | 1 | 0 | 1 |
| 33 | ON149707 | Penicillium rubens | 827 | 93 | 99.81 | FMR 13874 | 0 | 0 | 1 | 0 | 1 | 0 | 1 |
| 34 | ON149709 | Penicillium crustosum | 726 | 85 | 99.16 | FMR 1430 | 0 | 6 | 1 | 0 | 1 | 6 | 7 |
| 35 | ON149710 | Penicillium allii | 808 | 93 | 99.04 | FMR 14251 | 0 | 0 | 1 | 0 | 1 | 0 | 1 |
| 36 | ON149711 | Penicillium commune | 764 | 93 | 97.14 | CBS 311.48 | 0 | 0 | 1 | 0 | 0 | 1 | 1 |
| 37 | ON149712 | Mucor circinelloides f. circinelloides | 754 | 91 | 92.19 | IHEM 24129 | 1 | 0 | 0 | 0 | 0 | 1 | 1 |
| 38 | ON149713 | Geotrichum candidum | 482 | 90 | 99.03 | WM 07.304 | 1 | 0 | 0 | 0 | 0 | 1 | 1 |
| 39 | ON149714 | Geotrichum bryndzae | 436 | 82 | 98.93 | PMM09-440L | 1 | 0 | 0 | 0 | 0 | 1 | 1 |
| 40 | ON149715 | Fusarium keratoplasticum | 798 | 93 | 100.00 | FRC S-2465 | 0 | 1 | 0 | 0 | 0 | 1 | 1 |
| 41 | ON149716 | Penicillium brevicompactum | 738 | 93 | 97.67 | WM 06.340 | 0 | 5 | 2 | 0 | 0 | 7 | 7 |
| 42 | ON149717 | Alternaria alternata | 817 | 95 | 100.00 | WM 04.486 | 0 | 1 | 0 | 0 | 0 | 1 | 1 |
| 43 | ON149718 | Penicillium griseofulvum | 813 | 94 | 99.44 | CBS 185.27 | 0 | 0 | 1 | 0 | 0 | 1 | 1 |
| 44 | OM698376 | Penicillium palitans | 507 | 72 | 91.09 | FMR 14268 | 1 | 0 | 0 | 0 | 0 | 1 | 1 |
| Total | 55 | 55 | 66 | 72 | 36 | 68 | 176 | ||||||
The percentage of endophytic isolates belonging to Penicillium (59.66%; 105/176) was much higher than those identified as Aspergillus (23.29%; 41/176), Purpureocillium (6.25%; 11/176), Emericella (3.41%; 6/176), Fusarium (2.27%; 4/176), Paecilomyces (1.14%; 2/176), Geotrichum (1.14%; 2/176) as well as Altenaria, Microascus, Mucor, Pichia and Talaromyces, which were detected only sporadically (< 1%). The CR of the roots of the plant was higher (43.33%) than that of the stems (30.00%) and leaves (26.67%) of the identical plants. Furthermore, the CR of fungal endophytes of plants acquired from Loc-1 was much higher than that from Loc-2 and Loc-3.
To the best of our knowledge, this is the first report on the diversity of the endophytic fungi isolated from the invasive plant S. rostratum. This study demonstrated the comparatively high multiplicity of the endophytic fungi of S. rostratum from three locations in Xinjiang. Our work revealed that the invasive plant S. rostratum harbours a variety of fungal endophytes in its leaves, stems, and roots. Given the fact that endophytes are able to produce biologically active secondary metabolites that affect the growth of their hosts, we speculate that the endophytic fungi might contribute to the invasive success of S. rostratum.
Acknowledgments
This research work is financially supported by the National Foreign Experts Project granted to Nigora Kuchkarova (QN2022045006L), the CAS President’s International Fellowship Initiative (PIFI) granted to Zokir Toshmatov (2020PB0010), and the Natural Science Foundation of Xinjiang Uygur Autonomous Region (2022D01D02).
References
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Main Article Content
Abstract
A culture-dependent method was used to isolate fungal endophytes from the leaves, stems, and roots of the invasive plant Solanum rostratum Dunal. growing in Xinjiang Province, China. All isolates were identified according to ITS (internal transcribed spacer) region of ribosomal DNA sequences and analyzed by Nucleotide BLAST according to NCBI GenBank and Mycobank database. Altogether 176 endophytic fungal isolates corresponding to 44 OTUs were identified, which were classified into 12 genera, with Penicillium (59.66%) and Aspergillus (23.29%) being the highly dominant genera. Ten endophytic isolates (OTU1, OTU15, OTU16, OTU21, OTU23, OTU25, OTU26,
OTU30, OTU37 and OTU44) were identified as potential new species.
Keywords
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