Is allelochemical synthesis in Casuarina equisetifolia plantation related to litter microorganisms?

Abstract

Productivity decline of Casuarina equisetifolia plantation and difficulty in natural regeneration remains a serious problem because of allelopathy. Previous studies have confirmed that 2,4-di-tert-butylphenol (2,4-DTBP) are the major allelochemicals of the C. equisetifolia litter exudates. The production of these allelochemicals may derive from decomposition of litter or from the litter endophyte and microorganisms adhering to litter surfaces. In the present study, we aimed to evaluate the correlation between allelochemicals in litter and endophytic and epiphytic fungi and bacteria from litter. A total of 100 fungi and 116 bacteria were isolated from the interior and surface of litter of different forest ages (young, half-mature, and mature plantation). Results showed that the fermentation broth of fungal genera Mycosphaerella sp. and Pestalotiopsis sp., and bacterial genera Bacillus amyloliquefaciens, Burkholderia-Paraburkholderia, and Pantoea ananatis had the strongest allelopathic effect on C. equisetifolia seeds. Allelochemicals, such as 2,4-DTBP and its analogs were identified in the fermentation broths of these microorganisms using GC/MS analysis. These results indicate that endophytic and epiphytic fungi and bacteria in litters are involved in the synthesis of allelochemicals of C. equisetifolia. To further determine the abundance of the allelopathic fungi and bacteria, Illumina MiSeq high-throughput sequencing was performed. The results showed that bacterial genera with strong allelopathic potential were mainly distributed in the young and half-mature plantation with low abundance, while the abundance of fungal genera Mycosphaerella sp. and Pestalotiopsis sp. were higher in the young and mature plantations. In particular, the abundance of Mycosphaerella sp. in the young and mature plantations were 501.20% and 192.63% higher than in the half-mature plantation, respectively. Overall, our study demonstrates that the litter fungi with higher abundance in the young and mature plantation were involved in the synthesis of the allelochemical 2,4-DTBP of C. equisetifolia. This finding may be important for understanding the relationship between autotoxicity and microorganism and clarifying the natural regeneration problem of C. equisetifolia.

Keywords: 2; 4-DTBP; Casuarina equisetifolia; allelopathy; litter microbial community; microbial metabolites.

Figure 1

Effect of the 24 fungal strains on the germination of seeds of C. equisetifolia. Values marked with different letters are significantly different at p<0.05. (A) germination potential; (B) germination rate; (C) germination index.

Figure 2

The allelopathy index (resistance index) of the fungal strains. Values marked with different letters are significantly different at p<0.05.

Figure 3

Effect of the bacterial isolates on the germination rate of C. equisetifolia seeds. Values marked with different letters are significantly different at p<0.05. (A) germination potential; (B) germination rate; and (C) germination index.

Figure 4

The allelopathy index (resistance index) of the bacterial isolates. Values marked with different letters are significantly different at p<0.05.

Figure 5

Boxplots showing the Shannon and Simpson indices of the fungal and bacterial communities within different plantation ages. FY-1, FHM-1, FM-1 are the endophytic fungi of the young, half-mature, and mature plantation; FY-2, FHM-2, FM-2 are epiphytic fungi of the young, half-mature, and mature plantation; BY-1, BHM-1, BM-1 are endophytic bacteria of the young, half-mature, and mature plantation; BY-2, BHM-2, BM-2 are epiphytic bacteria of the young, half-mature, and mature plantation. The same bellows. (A) shannon index of OTU level of fungi; (B) simpson index of OTU level of fungi; (C) shannon index of OTU level of bacteria; and (D) simpson index of OTU level of bacteria.

Figure 6

Heatmap showing the dominant fungi and bacteria at the genus level in the litter of different plantation ages of C. equisetifolia. (A) community heatmap of fungi on family level; (B) OTU numbers of Mycosphaerella sp. and Pestalotiopsis sp.; (C) community heatmap of bacteria on genus level; (D) OTU numbers of Burkholderia-Paraburkholderia, Bacillus sp., and Pantoea sp.

Figure 7

Heat map showing the correlation of litter properties to the top 30 fungal and bacterial genera. X and Y axes represent the environmental factors and genera, respectively. R in different colors to show. *0.01< P ≤ 0.05, **0.001 < P ≤ 0.01,***P ≤ 0.001. (A) Fungi; (B) Bacteria.