<bold id="bold-76bbc2f013fd6a64385e6cb58fd4d04d">Introduction</bold>

Diversity of fungal endophytes isolated from the invasive plant Solanum rostratum

Kuchkarova

Nigora

nkn.bell.ixrv@mail.ru Han

Caixia

cxhani@ms.xjb.ac.cn Toshmatov

Zokir

zc@ms.xjb.ac.cn Chen

Hongyang

chenhongyang524@163.com Shao

Hua

shaohua@ms.xjb.ac.cn

State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China China State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China China State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China; Institute of Genetics and Plant Experimental Biology, Academy of Sciences of the Republic of Uzbekistan, Kibray, Tashkent region 111208, Uzbekistan China State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China China State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China; Research Center for Ecology and Environment of Central Asia, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China; University of Chinese Academy of Sciences, Beijing 100049, China China 10 01 2023 28 03 2024

2803202483

<bold id="bold-76bbc2f013fd6a64385e6cb58fd4d04d">Introduction</bold>

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.

<bold id="bold-6a335103fc968b9ede942a4c5fd7839f">Materials and methods</bold>

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.

<bold id="bold-5a12521d98b45132506aa4162f03dc1e">Results and discussion</bold>

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.

Table 1

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.

<bold id="bold-86906b692555bee59a2854d93df8a0fd">Acknowledgments</bold>

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).

<bold id="_bold-18">References</bold>

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		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