Anatomical, chemical and endophytic fungal diversity of a Qi-Nan clone of Aquilaria sinensis (Lour.) Spreng with different induction times

Abstract

Recently, some new Qi-Nan clones of Aquilaria sinensis (Lour.) Spreng which intensively produces high-quality agarwood have been identified and propagated through grafting techniques. Previous studies have primarily focused on ordinary A. sinensis and the differences in composition when compared to Qi-Nan and ordinary A. sinensis. There are few studies on the formation mechanism of Qi-Nan agarwood and the dynamic changes in components and endophytic fungi during the induction process. In this paper, the characteristics, chemical composition, and changes in endophytic fungi of Qi-Nan agarwood induced after 1 year, 2 years, and 3 years were studied, and Qi-Nan white wood was used as the control. The results showed that the yield of Qi-Nan agarwood continued to increase with the induction time over a period of 3 years, while the content of alcohol extract from Qi-Nan agarwood reached its peak at two years. During the formation of agarwood, starch and soluble sugars in xylem rays and interxylary phloem are consumed and reduced. Most of the oily substances in agarwood were filled in xylem ray cells and interxylary phloem, and a small amount was filled in xylem vessels. The main components of Qi-Nan agarwood are also chromones and sesquiterpenes. With an increasing induction time, the content of sesquiterpenes increased, while the content of chromones decreased. The most abundant chromones in Qi-Nan agarwood were 2-(2-Phenethyl) chromone, 2-[2-(3-Methoxy-4-hydroxyphenyl) ethyl] chromone, and2-[2-(4-Methoxyphenyl) ethyl] chromone. Significant differences were observed in the species of the endophytic fungi found in Qi-Nan agarwood at different induction times. A total of 4 phyla, 73 orders, and 448 genera were found in Qi-Nan agarwood dominated by Ascomycota and Basidiomycota. Different induction times had a significant effect on the diversity of the endophytic fungal community in Qi-Nan. After the induction of agarwood formation, the diversity of Qi-Nan endophytic fungi decreased. Correlation analysis showed that there was a significant positive correlation between endophytic fungi and the yield, alcohol extract content, sesquiterpene content, and chromone content of Qi-Nan agarwood, which indicated that endophytic fungi play a role in promoting the formation of Qi-Nan agarwood. Qi-Nan agarwood produced at different induction times exhibited strong antioxidant capacity. DPPH free radical scavenging activity and reactive oxygen species clearance activity were significantly positively correlated with the content of sesquiterpenes and chromones in Qi-Nan agarwood.

Keywords: Aquilaria sinensis; Qi-Nan; antioxidant activity; chemical composition; endophytic fungi.

Figure 1

(A–C) The agarwood yield of Qi-Nan. (D) The ethanol extract content of Qi-Nan. Different letters denote significant (P < 0.05) differences among the treatment in a one-way ANOVA and the bar represents standards deviation (n=3). 0a, before drilling; 1a one year after treatment; 2a, two years after treatment; 3a, three years after treatment.

Figure 2

Changes in starch and agarwood formation over time on cross-section (A–D). Changes is soluble sugar and agarwood formation over time on cross-section (E–H). IP, interxylary phloem; S, starch; F, xylem fiber; V, xylem vessel; XR, xylem ray; O, oil; SS, soluble sugar.

Figure 3

The changes of chemical composition of Qi-Nan before and after drilling for 1, 2, 3 years. (A) The component composition of white wood of Qi-Nan before drilling. (B) The component composition of Qi-Nan agarwood after 1 year drilling. (C) The component composition of Qi-nan agarwood after 2 years of drilling. (D) The component composition of Qi-Nan agarwood after 3 year of drilling.

Figure 4

Chao 1 (A) and ACE (B) index of Qi-Nan endophytic fungi communities with different induction time (n=3). 0a, before drilling; 1a; one year after drilling; 2a, two years after drilling; 3a, three years after drilling. Different letters denote significant (P < 0.05) differences among the treatment in a one-way ANOVA and the bar represents standard deviation (n=3).

Figure 5

ANOSIM (A) and PCoA (B) of Qi-Nan endophytic fungi in different induction time. 0a, before drilling; 1a one year after drilling; 2a, two years after drilling; 3a, three years after drilling.

Figure 6

The classification of Qi-Nan endophytic fungi communities at order levels (A) and genus levels (B) (relative abundance exceeded 1%) under different induction time and the relative abundance of each order and genus. 0a, before drilling; 1a; one year after drilling; 2a, two years after drilling; 3a, three years after drilling.

Figure 7

The relative abundance of Qi-Nan endophytic fungi communities at order levels (A) and genus levels (B) under different induction time. Different letters indicate significant differences under different induction time (P < 0.05), “NS” shows no significant difference. 0a, before drilling; 1a; one year after drilling; 2a, two years after drilling; 3a, three years after drilling. The abscissa in the figure corresponds to the order and genus of endophytic fungi in Figure 6 .

Figure 8

The phylogentic relationship of the top ten genera of endophytic fungi in Qi-Nan agarwood.

Figure 9

The co-occurrence network of Qi-Nan endophytic fungi communities at the order level in different induction time. Each node size is based on their relative abundance. The red and green edges represented the positive and negative correlation between Qi-Nan endophytic coefficient. (A) Before drilling; (B) one year after drilling; (C) two years after drilling; (D) three years after drilling.

Figure 10

Results of (A) DPPH free radical scavenging activity (SC) and (B) reactive oxygen species clearance (RC). Different letters denote significant (P < 0.05) differences among the treatment in a one-way ANOVA and the bar represents standard deviation (n = 3).

Figure 11

Correlation between components, active free radical scavenging ability and endophytic fungi of Qi-Nan agarwood. ***, P<0.001, indicates strong correlation.