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ALLELOPATHIC EFFECT OF CASTANOPSIS KAWAKAMII FOREST LITTER ON SEED GERMINATION OF SMALL PHILIPPINE ACACIA ( ACACIA CONFUSA)

. Nutrient limitation constrains the regeneration of Castanopsis kawakamii seedlings in subtropical forests. Small Philippine acacia ( Acacia confusa ), a common tree species in mixed forests, is known for its nitrogen fixation capabilities. Therefore, we attempted to intercrop A. confusa in a C. kawakamii natural forest. However, this forest contained abundant litter, harboring ample tannins and total phenols. Whether the allelopathy of litter could constrain the seed germination of A. confusa seed was unknown. Therefore, we aimed to explore the influence of forest litter extracts on the seed germination and radicle growth of A. confusa . The results indicate that low concentration litter extracts promote whereas high concentrations inhibit A. confusa seeds. The stock solution produced the strongest inhibition, whereas the 1:160 litter extract concentration had promoted the germination rate and radicle growth. The A. confusa radicle increase was remarkable in the first eight days, and tended to decrease from day 9 to 15. Overall, the allelopathic effect of litter is not a factor limiting the mixing of C. kawakamii and A. confusa . This could provide an alternative method to improving soil nutrition, thus promoting the regeneration of C. kawakamii seedlings.


Introduction
Castanopsis kawakamii is a unique subtropical evergreen broad-leaved species. It has survived as an endangered tertiary relict plant, only found in Fujian, Taiwan, Jiangxi, Guangdong, and Guangxi provinces in China (Buanjan et al., 2018). Currently, natural C. kawakamii forests are characterized by limited seed germination and seedling growth due to the low total nitrogen and total phosphorus in the soil . As crucial aspects of a forest ecosystem, the physical and chemical properties and nutrients in the soil are significant to seedling development (Hou et al., 2011). However, previous studies mainly focused on community ecology (He et al., 2019); forest gap regeneration ; natural forest, its plantation litter, and soil biochemistry cycle (Ma et al., 2015); soil microbes, biochemical characteristics (Ma et al., 2014); and soil-litter

Study sites and natural history
The C. kawakamii Nature Reserve is located in Sanming, Fujian Province, China, at 26°07'-26°12' N and 117°24'-117°29' E. The elevation varies between 180 and 604 m. The climate is medium subtropical monsoon, with an annual average temperature of 19.5 °C, annual precipitation around 1500 mm, and annual relative humidity of 79%. The soil type in this forest is mainly ferric acrisols with abundant humus. The research area has 700 ha of C. kawakamii, in which the average age of the population is about 130 years. The arbor layer is dominated by C. kawakamii, Pinus massoniana, and Schima superba. Accordingly, the population density, average tree height, diameter at

Preparation of litter extracts
Each kilogram of collected litter sample was washed three times with distilled water to remove surface-attached soil and humus. Next, the samples were crushed and sieved after drying in an oven. After sieving, 10 g of litter were placed into a 50 mL centrifuge tube and 15 mL of 70% ethanol was added. The mixture was leached twice at 80 °C for 1 h in a water bath. After free cooling, the samples were placed into a centrifuge at 4 °C for 5 min at a rotation speed at 8000 rpm. To obtain a stock solution (with three replicates), we filtered the extracts twice and then diluted the extracts to 50 mL with distilled water. As the extraction of litter in nature is repetitive and diverse, the range of extract types and contents is relatively wide. Ethanol was applied as an effective extraction agent to ensure polarized allelochemicals could be maximally extracted out of the litter.
The stock solution was then placed on a rotary evaporator to evaporate the ethanol. The average litter quality and annual precipitation of the C. kawakamii natural forest fixed plot from 2010 to 2017 were 11,010 kg/hm 2 and 1500 mm/year. Referring to the litter conversion (Yuan et al., 2010), 1:160 is the natural leaching concentration of the litter of natural C. kawakamii forest. Considering the rain leaching process, repeated extraction, and microbial decomposition, five concentrations were set as treatments: 1:20, 1:40, 1:80, 1:160, and 1:320. Therefore, the stock solution was diluted again with distilled water with five different concentrations. The dilution rates are listed as follows: 1:20 litter extracts (stock solution was diluted 4 times with distilled water), 1:40 litter extracts (stock solution was diluted 8 times with distilled water), 1:80 litter extracts (stock solution was diluted 16 times with distilled water), 1:160 litter extracts (stock solution was diluted 32 times with distilled water), and 1:320 litter extracts (stock solution was diluted 64 times with distilled water), and then we stored the extracts in a 4 °C refrigerator until use. During the extraction, the ethanol evaporation was incomplete. As a consequence, we also created a blank control group as a contrast that was exempted from the treatment: the same procedure was repeated but without the litter.

Seed germination experiment
The selected and same-sized A. confusa seeds were disinfected with 0.5% potassium permanganate for 30 min, then washed with distilled water 3 times before arranging them evenly in a petri dish with C. kawakamii forest soil. The C. kawakamii forest soil was sterilized in a high temperature sterilization pot at 120 °C for 20 min. The different concentrations of litter extracts were added into each petri dish, with 100 seeds placed uniformly in each petri dish. We set up a total of 6 treatments including stock solution, 1:20 litter extract, 1:40 litter extract, 1:80 litter extract, 1:160 litter extract, and 1:320 litter extract with 3 replications each with distilled water, and a blank control group as the control. The experiment conditions were cultured with a 12 h photoperiod at 25 °C, 12 h dark cycle at 15 °C, light intensity 50 nmol/cm 2 /s, and relative humidity 75-80% in the incubators. We added 5 mL litter extract daily into the filter paper of the petri dish to maintain moisture.
We recorded the seed germination 3 days after the experiment. A 2 mm seed radicle was defined as seed germination, and the number of germinated seeds was recorded every day (Aldo et al., 2018). We finished the experiment when no further seed germination occurred for 3 continuous days. The radicle length measurements were recorded when the seed germination reached the peak (Wang et al., 2019b). We measured the radicle length of 20 seeds with a similar radicle length in different petri dishes after recording the seed germination for three days when the seed germination reached the peak. The length of the radicle was also measured daily with an electronic digital caliper with a resolution of 0.01 mm. The radicle daily growth amount was simultaneously calculated by the difference in the value of the radicle length between the current day and the previous day. On the 15th day, we finished the radicle experiment when the radicle growth was generally stable. Finally, the germination rate, germination potential, germination index, allelopathic effect index, and the radicle length of seeds were thereby calculated (Bruce et al., 1988 where Dt refers to the number of germination days, and Gt refers to the number of germinated seeds in that Dt day. Allelopathy index: where C refers to the control value, T refers to the treatment value, while I < 0 represents the inhibition effect, I > 0 represents the promotion effect, and I = 0 represents the neutral effect.

Data analysis
The experimental data were calculated and plotted using Excel 2016 (Microsoft, Redmond, Washington, USA). The statistical ANOVA was analyzed using SPSS 21.0 (IBM, Almonk, New York, USA). We first calculated the effect of different concentrations of litter extracts on the germination indices, radicle length, radicle growth, and allelopathy index of A. confusa seeds. Then, the variance analysis of these data was performed, and Tukey's test was applied in multiple comparisons to determine the significant difference among various treatments. The data were considered to be significantly different at P < 0.05.

Effects of different extract concentrations on germination of A. confusa seeds
The effects of various concentrations of litter extracts on seed germination of A. confusa is shown in Tables 1 and 2. A. confusa seeds demonstrated different sensitivities to different concentrations of litter extract, which generally promoted seed germination at low concentrations and inhibited it at high concentrations. We found a negative correlation between litter extract concentration and germination potential, germination rate, and germination index. The germination rate was highest with a litter concentration of 1:160, which promoted the seed germination rate. The seed germination rate was significantly lower than those of the other treatments at a concentration of 1:20, demonstrating the most inhibition of the seed germination rate. The seed germination rates under high concentration treatments (including stock solution, 1:20, and 1:40) were significantly lower than under low concentration treatments (1:160 and 1:320). High concentrations of litter extract inhibited the seed germination rate, whereas low concentrations had a promotion effect. The germination potentials for high concentration (including stock solution and 1:20) flitter extract treatments were significantly lower than for low concentration (1:80 and 1:160) treatments. The germination potential and index were the lowest for the stock solution, which revealed negative effects on the seed germination potential and index. The germination indexes for the high concentration (including stock solution and 1:20) treatments were significantly lower than for the low concentration litter extract treatments (including 1:80, 1:160, and 1:320). No significant differences in germination rate, potential, or index were found between distilled water and the control group. We found that when the concentration of litter extracts exceeded the dilution ratio of 1:40, the seed germination was restrained. The 1:80 dilution rate was considered the turning point for seed germination; the optimum dilution for seed germination was found to be 1:160. The 1:160 litter extract was the most appropriate concentration for the germination of A. confusa seeds.

Effects of different concentrations of extracts on radicles of A. confusa seeds
Difference in radicle length between different concentrations of extracts Figure 2 and Table 3 show the effects of various concentrations of litter extract on the radicle length of A. confusa seeds. Similar to seed germination, the different concentrations of litter extracts tended to promote radicle growth at low concentration and inhibit radicle growth at high concentration. The inhibition effect was aggravated with increasing concentration, among which the most notable effect was observed with the stock solution. The 1:40 to 1:160 litter extracts demonstrated a promotion effect, that was the strongest at 1:160, whereas the facilitation effect gradually weakened when the concentration was 1:320. The growth in radicle length in the early stage was not obvious for the different concentrations of litter extracts. The difference in radicle length between various concentration of litter extracts and distilled water increased with time. Significant differences in radicle length were detected among the stock solution, distilled water, and 1:160 concentration litter extract. At the end of the experiment, the average radicle lengths for 1:160 litter extract, distilled water, and stock solution were 36.09, 30.06, and 19.59 mm, respectively. No significant differences in radicle length were found between the distilled water and control groups. We found that A. confusa radicle elongation was inhibited under the stock solution and 1:20 litter extract; at 1:40, 1:80, and 1:160 litter extracts, we observed promotion effects, although it was not conspicuous at the concentration of 1:320 ( Table 3). The stock solution had the strongest inhibiting effects on seed radicle growth. The best promotion effect was detected for the 1:160 litter extract which showed a relative high promotion effect in the early period, then later decreased. Consequently, the 1:160 litter extract remained the optimum concentration for the growth of A. confusa radicle length.
The results of the two-way ANOVA analysis of variance were presented in Table 4. There was a significant difference in the length of A. confusa radicle treated with time or different concentrations of litter extracts. But the difference in interaction between time and different treatments is not significant. Day  D3  D4  D5  D6  D7  D8  D9 D10 D11 D12 D13 D14

Differences in radicle growth of A. confusa seeds in different time periods
The effects of litter extracts on the seed radicle growth is shown in Figure 3. It shows that the seed radicle growth of A. confusa decreased with time, and the growth trends of various concentrations were different. The growth of the seed radicle was greater in the distilled water group at the period from day 4 to 9, and the growth was stable after the 10th day. The radicle growth increased the most on the fifth day and the least on the 11th day. For the stock solution of litter extracts, the seed radicle growth during day 5 to 7 was significantly higher than on other days; the seed radicle grew the most on the fifth day and the least on the 13th day. The 1:20 litter extract group demonstrated the most seed radicle growth on the fifth day and the least on the eighth day; the seed radicle growth during the first eight days was relatively high compared to that on the following days. At the 1:40 concentration, the radicle growth was relatively high during day 4 to 10 (except for the eighth day); the maximum growth occurred on the fourth day, with the least growth on day 11 to 14, and the radicle growth gradually increased in the later stage. At 1:80 and 1:160 concentrations, the seed radicle growth showed a general downward trend, reaching the maximum on the fourth day, and stabilized after decreasing sharply on day 10 to 15. The seed radicle growth at the 1:320 concentration reached a maximum on the sixth day, then declined from day 7 to 16, and the least growth was observed on the 16th day. The different concentrations of litter extracts inhibited the seed radicle growth less in the early period, where the radicle growth was relatively fast, whereas the various concentrations of litter extract had different effects on seed radicle growth in the later stages. Compared with the distilled water group and the control group, the high litter extract concentrations inhibited the radicle growth of A. confusa, which was promoted at low concentrations. http The A. confusa seed radicle growth was inhibited on day 7 to 9 and day 12 to 15, and promoted on day 5 to 6 and 10 to 11 in general ( Table 5). The inhibition effect was obvious in the stock solution and the 1:20 concentration litter extract. The promotion effect appeared in the 1:80, 1:160, and 1:320 litter extract concentrations. The different days and treatments, along with their interaction, significantly affected the radicle growth of A. confusa (Table 6). Overall, the 1:160 litter extract concentration was the optimum for the increase in A. confusa radicle length.

Discussion
With increasing litter extract concentration, the germination potential, germination rate, and germination index of A. confusa decreased. The stock solution and the 1:20 litter extracts concentrations had strongest inhibitory effect on seed germination. The germination potential and germination index of the seeds in the stock solution were the lowest, primarily due to the restraining effect of the allelochemicals in the stock solution on the speed of seed germination. The germination rate in the 1:20 litter extraction concentration group was the lowest and the negative effect of allelochemicals on the seeds were most pronounced. This may be caused by the amount of harmful ingredients in the allelochemicals, which were much more than the amount of beneficial components. Under normal circumstances, the production and elimination of free radicles in plant cells maintains a dynamic equilibrium; under stress conditions, this balance is affected and readily destroyed. When free radicals accumulate to a certain extent, membrane lipid peroxidation occurs, which inhibits seed germination (Shibaeva et al., 2018;Marusa and Ishii-Iwamoto., 2011). At the 1:160 concentration, the three seed germination indices were the highest, and the promotion effect was the most obvious. We speculate that this concentration is the optimum concentration of seed germination, and the beneficial components in the allelochemicals accumulate the most. Within this concentration range, the ion transporters in the cell and various ion channel proteins are involved in cell ion homeostasis, increasing seed germination rate and germination speed (Steven et al., 2003;Zhang et al., 2016;Bachleda et al., 2017;Kilic et al., 2016). The 1:320 litter extract concentration also promoted the germination of seeds, but the overall effect was not significant. The reason for this may be that the concentration was lower and similar to distilled water, and the allelochemicals content was relatively low; thus, the promoting effect was not obvious. In general, compared with the distilled water group, no germination index was significant for the lowconcentration litter extracts, whereas high-concentration litter extracts significantly inhibited the germination indices of A. confusa seeds. When the allelochemicals content is limited, the plant itself produces stress-resistant immunity. At this time, allelopathic chemicals have little effect on the plant. When the residual allelochemicals increase, accumulate, and increase to a certain amount or exceed the critical content, they are harmful to the plant and thus delay seed germination (Liu et al., 2016;Wei et al., 2018).
With the increase in litter extract concentration, the seed radicle growth of A. confusa showed an obvious hysteresis effect. The litter extracts had a significant inhibiting effect on seed radicle growth compared with distilled water, mainly because the seed radicle growth was inhibited by the stock solution and the 1:20 concentration. High litter extract concentrations may have physiologically disturbed the plant, inducing intensive anaerobic respiration, which would therefore weaken the plant's ability to scavenge free radicals, resulting in a decrease in its physiological index (Steven et  and enzyme activity in the seeds of A. confusa may have led to the inhibitory effect of litter extracts on seed germination and radicle growth within a certain concentration range (Hunter et al., 2013). When the litter extract concentration was 1:160, the promotion of radicle growth was the most obvious, and the content of beneficial allelochemicals was the highest. We speculate that the concentration of nutrients in this treatment was high, the breathing was strong, the high energy bond in ATP was active, the mitochondria inner membrane activity was strong, the rates of electron transfers and oxidative phosphorylation were fast, and the cell division speed of the apical meristem was accelerated, which effectively promote radicle elongation (Steven et  By analyzing the growth in the radicle over time, we discovered that the radicle growth of A. confusa treated with litter extract was relatively high in the early stage, and the growth the middle and late stages tended to decline. The reason for this may be that a certain amount of allelopathic substances accumulated in the litter, hindering the water absorption of the seed; inhibiting the respiration, enzyme activity, and radicle growth; and changing the osmotic pressure, thereby constraining the elongation of the radicle (Wei et al., 2018). When the litter extract concentrations were 1:40 and 1:80, the seed radicle growth of A. confusa increased gradually in the later stage, which may be due to the increase in peroxidation products in the plant after the stress due to allelopathic substances. When these allelopathic substances reach a certain amount and the stress mechanism is constrained by the plant, its own protective enzyme system could thereby scavenge the free radicals generated. However, this adaptive response is effective, as the degree of damage was relatively low and reversable, whereas the radicle increase was limited at any higher concentration of litter extract (Marusa and Ishii-Iwamoto, 2011).
The low litter extract concentration in nature can promote the germination and radicle growth of A. confusa to some extent. So, it is feasible to mix A. confusa in C. kawakamii forests. However, when mixing A. confusa, we should try to select a location where the amount of litter is low and the topography is relatively flat to mitigate the adverse effects of allelopathy. To afforest a successful mixed forest, further studies are required. Firstly, the effects of extracts from C. kawakamii natural forest on A. confusa should be studied from the perspective of the physiological activity of the radicle. Secondly, the different effects of various types and contents of litter allelochemicals at different forest ages and stages on seeds should be thoroughly considered during the afforestation of mixed forests (Hunter et