OTU taxonomic status identification and sequencing data processing:
The sequence data, rigorously quality-controlled, reveal the diversity of bacterial and fungal endophytes in Zea mays seeds from various sites, including HM-contaminated locations (SD and AK) and a non-contaminated site (DL) in Yunnan Province, China (Table 1). Of the nine bacterial samples, approximately 492,177 high-quality reads were obtained, while nine fungal samples produced 1,001,229 optimized sequences, offering a comprehensive view of the microbial diversity influenced by HM stress.
Microbial diversity analysis:
The rarefaction curves in Fig. 1 show how the three regions' sequencing outcomes were determined. Each sample's curve hits a plateau, demonstrating that the sequencing adequately captured the samples' variety. The Chao1, ACE, Shannon, and Simpson diversity indices were analyzed. The diversity of bacteria and fungi found in maize seeds from the AK, DL, and SD regions is shown in Table 2. The fungal and bacterial richness was highest in the AK samples and lowest in the DL samples. According to the Shannon Index, the bacterial samples from AK and SD, and fungal samples from AK showed more diversity and evenness, respectively. In contrast, DL showed a more uniform distribution of fungal species, according to the Simpson Index. While DL and SD displayed a more uniform distribution of bacterial taxa, AK had a more dominant bacterial population. Finally, it can be said that the three study sites have different fungal and bacterial diversity in maize seeds.
Table 2. The first column is the sample name, and the remaining columns are the values corresponding to the diversity index types in each sample
Bacteria
|
sobs
|
Shannon
|
Simpson
|
ace
|
Chao
|
coverage
|
A1
|
189
|
2.028157
|
0.330451
|
192.848083
|
190.956522
|
0.999792
|
A2
|
68
|
1.024694
|
0.625476
|
75.671166
|
77.333333
|
0.999885
|
A3
|
100
|
1.849471
|
0.315525
|
158.238035
|
121
|
0.999598
|
S4
|
79
|
1.018447
|
0.628111
|
85.761863
|
82.928571
|
0.999828
|
S5
|
93
|
2.22932
|
0.207997
|
105.795221
|
101.666667
|
0.999775
|
S6
|
96
|
1.712651
|
0.402934
|
110.742832
|
104.571429
|
0.999736
|
D10
|
115
|
1.506716
|
0.526165
|
120.517448
|
120.5
|
0.999695
|
D11
|
95
|
1.564256
|
0.505741
|
106.704178
|
108
|
0.999649
|
D12
|
85
|
1.340296
|
0.514775
|
101.445749
|
107.666667
|
0.99953
|
Fungi
|
|
|
|
|
|
|
A1
|
92
|
1.326334
|
0.538526
|
96.321065
|
97.142857
|
0.999882
|
A2
|
79
|
2.01493
|
0.23574
|
91.913265
|
96.5
|
0.999848
|
A3
|
90
|
2.041316
|
0.244605
|
97.268603
|
96.428571
|
0.999884
|
S4
|
60
|
0.124184
|
0.961758
|
94.254775
|
83.214286
|
0.999835
|
S5
|
109
|
1.307874
|
0.406762
|
121.091275
|
121
|
0.999859
|
S6
|
75
|
1.094398
|
0.54845
|
81.631611
|
79.5
|
0.999882
|
D10
|
44
|
0.786603
|
0.494409
|
87.156318
|
59
|
0.999898
|
D11
|
67
|
0.331486
|
0.884503
|
109.319918
|
84.5
|
0.999832
|
D12
|
76
|
1.634705
|
0.318383
|
87.139166
|
86.5
|
0.999857
|
Note: A1-A3 = AK; S4-S6 = SD; D10-D12 = DL
Microbial community structure:
The NMDS analysis based on the unweighted unifrac distance showed. The findings show that samples from HM-contaminated areas have greater diversity than samples from uncontaminated areas (Fig. 2). A clustering tree analysis showed that the diversity of endophytic bacteria and fungi was remarkably high in the HM-contaminated areas (Fig. 4A and 4B). Additionally, the PLS-DA discriminant models for each region suggest that both models offered reliable diversity classification, confirming the variations in microbial communities within each location (Fig. 4C and 4D).
Microbial community composition:
The microbial community’s composition was assessed in all the samples collected from the selected regions. Fig. 4A demonstrate that the bacterial taxa are predominantly classified into the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidota, and Gemmatimonadota. The predominant bacterial taxa identified were unclassified_f__Alcaligenaceae, Pseudomonas, unclassified_p__Proteobacteria, Saccharopolyspora, and Gordonia (Fig. 4C). Majority of the fungal flora from the sample were classified within the phyla Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, and unclassified_k__Fungi (Fig. 4B). Further analysis revealed that the Unclassified_p__Ascomycota, Fusarium, Saccharomycopsis, Neofusicoccum, and Sarocladium were the top five dominating fungal genera (Fig. 4D).
Differential taxonomic composition analysis:
The Metastats approach was used to identify taxa with substantial differences across three distinct regions (Fig. 5). The abundant bacterial species found in AK seeds were Gordonia, Branchiibius, Burkholderia-Caballeronia-Paraburkholderi, Comamonas, and Microbacterium whereas, in the case of seeds from the SD, the dominant species were Pseudomonas, Saccharopolyspora, Bacillus, Brevundimonas, Chryseobacterium, Thauera, Streptomyces, Herbaspirillium, Massilia, and Lactobacillus. Among the three regions, the abundance of bacterial species like Rhodococcus and unclassified_f_Alcaligenaceae are uniformly distributed.
In case of fungal endophytes, Thermoascus, Aspergillus, Cutaneotrichosporon, Trichsporon, Cladosporium, Penicillium, and Talaromyces were more prevalent in seeds of the AK region when compared to those from the other two regions. The fungal endophytes like Fusarium, Saccharomycopsis, Lecanicillium, and Wickerhamomyces were more prevalent in SD seeds. Meanwhile, Neofusicoccum, Sarocladium, Nigrospora and unclassified_o_Sordariales were found in more abundance in non-contaminated region DL.
The LEfSe analysis identified the key bacterial and fungal taxa characterizing the three groups. Significant changes were recorded in the abundance of Bdellovibrionota, Bdellovibrionia, and Enterococcus (Fig. 5A). Peptostreptococcales-Tissierellales and Rhizobiacoae exhibit an increase in relative abundance in the AK samples as compared to other categories. The analysis also revealed the fungi with the highest mean abundance within each category. The top five fungi, demonstrating the different fungal composition at each location, were Capnodiales, Chaetothyriates, Cladosporiaceae, Trichomeriaceae, Cladosporium, and Knufia (Fig. 6B).