Lily (Lilium spp.) root exudates exhibit different allelopathies on four vegetable crops

Abstract

Lilies are a popular ornamental and vegetable crop that has medical use. Lily cultivation has developed quickly in China but faces continuous cropping obstacles. Allelopathy is one of the main continuous cropping obstacles and is the foundation for after-crop selection in crop rotation. In this study, lily root exudates were acquired at three stages (10, 20, and 30 days) after hydroponic culture of lily bulbs, and the allelopathy of four vegetable crops was tested by bioassay. The results indicated that the allelopathy intensity of lily root exudates on radish increased with increasing duration of hydroponic culture, and the lily root exudates that were collected after 30 days of hydroponic culture exhibited the strongest allelopathic effects. The allelopathy on tomato, cucumber, and lettuce was complex. The weakest allelopathy on tomato and cucumber, but the strongest allelopathy on lettuce, was exhibited by lily root exudates that were collected after 20 days of hydroponic culture. Furthermore, differences in seed germination response and shoot and root growth patterns of the same root exudates were observed. Comparably, radish was the most sensitive receptor to the allelopathy of lily root exudates that were collected at different stages, and lettuce appeared to be the least sensitive of the tested receptors based upon the studied indices. It is inferred that lettuce and cucumber are suitable after-crops for lily, while radish is the most rational receptor for further bioassay tests on the allelopathy of lily root exudates.

Keywords:

• Allelopathy
• hydroponic culture
• lily
• root exudates
• vegetable receptor

Introduction

Plant allelopathy is a phenomenon of donor plants that affects the growth and development of itself or the surrounding organisms by releasing allelochemicals to the environment (Williamson & Richardson, 1988). A donor plant releases allelochemicals in a variety of ways, such as leaching, volatilizing, decomposing of stubble, and root exudation. Allelopathy caused by root exudates is the main factor that leads to continuous cropping obstacles (Zhang et al., 2007; Liu et al., 2010). At present, the research on allelopathy of ornamental plants is mainly focused on the chrysanthemum family (Chon & Nelson, 2011), turf grasses and various tree species. However, more research has investigated the allelopathy of vegetable and other crops (Jilani et al., 2008; Kato-Noguchi et al., 2012), but only a few reports have been found on the allelopathy of lily (Lilium. spp) root exudates (Dong et al., 2008; Xu et al., 2011).

Lily is a popular ornamental and vegetable crop and is an important herb in the Chinese traditional medicine system. Because of its increasing market demand and good economic returns, large-scale cultivation in open fields, greenhouses, and plastic tunnels are gaining popularity in China. The statistics indicate that the production of lily bulbs for vegetable consumption, food processing, and medicinal uses has reached over 100,000 tons, 4000 tons and 4200 tons, respectively, in China (Ding, 2011). This large-scale, successive cultivation of lily has brought an ecological and agronomic problem because it has resulted in increased disease and pest attacks, deterioration of the physical, chemical, and biological properties of soil, suppression of plant growth and a reduction in yield and quality of crop production (Yu et al., 2004). Continuous cropping or monoculture leads to the loss of biodiversity in agro-ecosystems (Mediene et al., 2011), and increasing vegetation biodiversity can reduce the impact of diseases and pests (Ratnadass et al., 2012). Suitable after-crop selection may be an effective strategy for coping with this problem by encouraging flora diversity in agro-ecosystems.

In this study, four typical vegetable crops, cucumber, lettuce, tomato, and radish, were selected as receptors to investigate the allelopathy of lily root exudates. The results from this study may provide a theoretical basis to select reasonable after-crops and develop a rational farming system. They may also be useful for screening the most sensitive receptor to be used for further research on identifying allelochemicals in lily root exudates.

Materials and methods

Experimental materials

Donor plant: bulbs of Siberian lily (Lilium spp.) were purchased from Jiangsu Shuyang MLC Field Flower Gardening (Shuyang County, Jiangsu Province, China).

Receptor plant: Seeds of radish (Raphanus sativus), cucumber (Cucumis sativus), lettuce (Lactuca sativa) and tomato (Lycopersicon esculentum) were procured from Nongcheng Seed Technology Center of Northwest A & F University.

Collection of lily root exudates

Lily root exudates were collected by hydroponic culture of lily bulbs (Xu et al., 2011). A total of 300 uniformly sized lily bulbs were soaked in 0.1% carbendazim for half an hour, rinsed several times with tap water, and then sowed in sterilize, moistened perlite. After rooting, the bulbs were gently removed from the soil while avoiding any root damage, rinsed to remove perlite particles and then transplanted on a perforated thermopore plate that allowed seedling growth on a self-designed, simple hydroponic culture system that had continuous ventilation that was facilitated by electric ventilators. In each system, 12 lily bulbs were arranged in a square layout (5×5 cm), and 1500 ml of distilled water was added into a flat-surfaced, 6000 ml capacity, rectangular, plastic tub. Because rich nutrients are present in lily bulbs, no exogenous nutrition was supplied. The experiment was conducted at room temperature, the water level was adjusted to 1500 ml per tub during culturing, and three 400 ml culture solutions were sampled every 10 days. The sampled culture solution with lily root exudates was stored in a refrigerator at (4°C) for further bioassay studies.

Bioassay of the allelopathy of lily root exudates

The allelopathy of lily root exudates on the four receptors was tested using a bioassay method (Williamson & Richardson, 1988). Seeds of the receptors were first sterilized in 0.3% potassium permanganate solution for 10 min and then rinsed 2–3 times with sterilized, distilled water. About 100 seeds of each receptor were sown in culture dishes (150 mm), which each contained a filter-paper layer. Initially, 8 ml of lily root exudates, which had been collected at different stages of hydroponic culture, were added to their respective culture dishes, and 2 ml of the same root exudates were poured into the culture dish every 2 days. For the control, only distilled water was added using the same pattern. The dishes were incubated at 25°C, and the experiment was replicated thrice. The number of germinated seeds in each dish was counted daily, and the root length, shoot length, fresh weight of root, and fresh weight of shoot were measured after 10 days of incubation.

Data processing and statistical analysis

The allelopathic reaction index (RI) was adopted to analyze the allelopathy of lily root exudates on the four receptors (Williamson & Richardson, 1988) using the following formulae:

Here, RI refers to the extent or intensity of allelopathy, T stands for the treatment value, and C stands for the control value for an investigated parameter. An RI value > 0 refers to a stimulatory effect and vice versa. All data were processed, and analysis of variance was computed using the DPS (Version 7.5) software.

Results

Allelopathy of lily root exudates that were collected after 10 days of hydroponic culture on different receptors

Lily root exudates that were collected after 10 days of hydroponic culture presented different allelopathic effects on different receptors and on different growth indices (Table I). A significant (P≥95%), positive allelopathic effect on the germination rate, root length, shoot length, root fresh weight, and shoot fresh weight of radish was observed, and the promotion rates were found to be 12.7%, 47.5%, 18.4%, 31.9%, and 12.3%, respectively. In contrast, no significant (P≥95%) effect was observed on any indices of lettuce and cucumber or on any other index except for root fresh weight of tomato. However, small inhibitory effects on the germination rate, root length, shoot length and shoot fresh weight of lettuce, on the germination rate, shoot length and shoot fresh weight of tomato, and on the germination rate of cucumber were found. However, only the inhibitory effect on root fresh weight of tomato was significant (P≥95%). Furthermore, small, positive effects on the root fresh weight of lettuce, on the root length of tomato, and on all indices except for the germination rate of cucumber were found (Table I).

Table I. Effect of lily root exudates collected after 10 days of hydroponic culture on seed germination and shoot and root growth of different receptors.

Receptor	Germination rate (%)	Root length (cm)	Shoot length (cm)	Root fresh weight (mg)	Shoot fresh weight (mg)
Radish control	81.33 a	2.61 a	4.62 a	7.06 a	73.18 a
Radish	91.67 b	3.85 b	5.47 b	9.31 b	82.19 b
Lettuce control	91.66 a	4.03 a	2.83 a	3.11 a	13.10 a
Lettuce	91.33 a	4.00 a	2.67 a	3.94 a	12.51 a
Tomato control	91.00 a	5.81 a	4.45 a	4.03 a	18.21 a
Tomato	83.67 a	5.91 a	3.96 a	2.43 b	16.13 a
Cucumber control	96.67 a	6.26 a	4.41 a	16.65 a	86.57 a
Cucumber	93.33 a	6.62 a	4.70 a	26.36 a	101.92 a
Note: Different small letters indicate significant differences at 5% level by Duncan's test.

Allelopathy of lily root exudates that were collected after 20 days of hydroponic culture on different receptors

Lily root exudates that were collected after 20 days of hydroponic culture presented different inhibitory allelopathic effects on all of the indices of radish, lettuce, and tomato, and on most indices of cucumber.

Inhibition of the germination rate, root length, shoot length, root fresh weight, and shoot fresh weight of radish was 17.9%, 42.2%, 16.0%, 31.2%, and 6.2%, respectively; however, only the germination rate and root fresh weight were significantly inhibited (P≥95%). Inhibition of the germination rate, root length, shoot length, root fresh weight, and shoot fresh weight of lettuce was 3.2%, 14.1%, 20.1%, 5.0%, and 1.6%, respectively, but no significant (P≥95%) inhibition was recorded. Inhibition of the germination rate, root length, shoot length, root fresh weight, and shoot fresh weight of tomatoes was 4.5%, 10.2%, 4.2%, 29.1%, and 5.9%, respectively, but as before, these effects were not significant (P≥95%). Lily root exudates exhibited 17.6%, 5.5%, and 0.6% inhibition on the root length, shoot length and shoot fresh weight of cucumbers, respectively, but showed a 1.2% and 4.1% promotion to germination rate and root fresh weight, respectively. However, no significant (P≥95%) inhibition or promotional effects were documented (Table II).

Table II. Effect of lily root exudates collected after 10 days of hydroponic culture on seed germination and shoot and root growth of different receptors.

Receptor	Germination rate (%)	Root length (cm)	Shoot length (cm)	Root fresh weight (mg)	Shoot fresh weight (mg)
Radish control	76.33 a	2.82 a	4.95 a	6.77 a	49.92 a
Radish	62.67 b	1.63 a	4.16 a	4.66 b	46.82 a
Lettuce control	93.00 a	4.90 a	3.44 a	4.21 a	13.39 a
Lettuce	90.00 a	4.21 a	2.75 a	4.00 a	13.18 a
Tomato control	86.00 a	7.19 a	5.05 a	4.75 a	20.73 a
Tomato	83.00 a	6.46 a	4.89 a	3.37 a	19.50 a
Cucumber control	94.00 a	6.71 a	4.54 a	18.35 a	96.03 a
Cucumber	96.00 a	5.53 a	4.29 a	19.11 a	95.38 a
Note: Different small letters indicate significant differences at 5% level by Duncan's test.

Allelopathy of lily root exudates that were collected after 30 days of hydroponic culture on different receptors

Lily root exudates that were collected after 30 days of hydroponic culture showed similar levels of inhibition to those of the 20-day-culture exudates on radish and tomato but exhibited different levels of promotion on lettuce. The inhibition rates of radish seed germination, root length, shoot length, root fresh weight, and shoot fresh weight were 27.1%, 42.2%, 25.3%, 38.3%, and 15.6%, respectively. All of these inhibition effects were significant (P≥95%) except for shoot fresh weight. The promotion rates of lettuce seed germination, root length, shoot length, root fresh weight, and shoot fresh weight were 1.8%, 2.7%, 8.9%, 1.8%, and 14.6%, respectively, but none of the promotional effects were statistically significant (P≥95%). The inhibitory effects on the germination rate, root length, root fresh weight, and shoot fresh weight of tomato were 4.5%, 16.4%, 29.7%, and 5.9%, respectively, but a 4.2% promotion of shoot length was observed. However, neither the inhibition nor promotion was statistically significant (P≥95%). Lily root exudates showed a 2.1% promotion to the germination rate but a 4.3%, 11.7%, 28.3%, and 6.6% inhibition of root length, shoot length, root fresh weight, and shoot fresh weight, respectively, of cucumber. However, statistically, this promotion was not significant (P≥95%) (Table III).

Table III. Effect of lily root exudates collected after 10 days of hydroponic culture on seed germination and shoot and root growth of different receptors.

Receptor	Germination rate (%)	Root length (cm)	Shoot length (cm)	Root fresh weight (mg)	Shoot fresh weight (mg)
Radish control	82.33 a	3.27 a	5.81a	7.67 a	67.69 a
Radish	60.00 b	1.89 b	4.34 b	4.73 b	57.16 a
Lettuce control	92.67 a	3.75 a	3.15 a	3.87 a	14.97 a
Lettuce	94.33 a	3.85 a	3.43 b	3.94 a	17.15 a
Tomato control	86.00 a	6.17 a	6.13 a	4.78 a	20.73 a
Tomato	83.00 a	5.16 a	6.39 a	3.36 b	19.50 a
Cucumber control	94.00 a	6.26 a	6.44 a	28.34 a	125.64 a
Cucumber	96.00 a	5.99 a	5.69 a	20.33 b	117.31 b
Note: Different small letters indicate significant differences at 5% level by Duncan's test.

Comparison of the RI of lily root exudates that were collected at different culture stages

From the RI data, it was established that radish was the most sensitive and lettuce was the least sensitive receptor of the four studied receptors. Lily root exudates that were collected at any stage of hydroponic culture showed inhibitory allelopathic effects on tomato, but negligible differences in RI values between all three stages were observed.

Lily root exudate samples that were collected after 10 days of hydroponic culture showed positive (promotion) allelopathic effects on radish, lettuce, and cucumber, those collected after 20 days of hydroponic culture showed negative (inhibitive) allelopathic effects on all receptors, and those collected after 30 days of hydroponic culture showed negative (inhibitive) allelopathic effects on radish and tomato but positive (promotion) allelopathic effects on lettuce and cucumber (Figure 1). The absolute value of RI increased markedly for radish and slightly decreased for tomato with an increase in duration of lily hydroponic culture. For cucumber, the highest absolute value of RI was exhibited by lily root exudates that were collected after 10 days of hydroponic culture, and the lowest absolute value of RI was presented by lily root exudates that were collected after 20 days of hydroponic culture. However, for lettuce, the lowest absolute value of RI was exhibited by lily root exudates that were collected after 10 days of hydroponic culture, and the highest absolute value of RI was presented by lily root exudates that were collected after 20 days of hydroponic culture.

Figure 1.  Comparison of RI of lily root exudates collected at different culture stages.

Discussion

The selectivity of the allelopathy of lily root exudates to receptors and growth indices

Allelopathy has selectivity for receptor plants, i.e., allelopathic effects are different for different receptor plants. Zheng and Feng (2005) indicated that Trifolium repens, rhodes grass, Mariscus cyperinus, and Ixeridium gracile were sensitive to the allelochemical in Eupatorium adenophorum extracts; conversely, Medicago sativa was insensitive to this chemical. Li et al. (2010) reported that the inhibition effect of the allelochemical in Artemisia vulgaris extract on rape germination speed and seedling growth was stronger than on those of cucumber, sorghum, radish, and wheat. Zhou et al. (2007, 2011) investigated the allelopathy of garlic root exudates and found that tomato and radish were sensitive but cucumber was relatively less sensitive to garlic root exudates. Furthermore, the root exudates of two garlic cultivars exhibited positive effects on seed germination and seedling growth of lettuce at low concentrations (0.1 and 0.2 g·mL⁻¹) but exhibited inhibitory effects at high concentrations (0.4 and 0.6 g·mL⁻¹). Garlic root exudates also showed inhibitory effects on the mycelia growth and spore germination of the pathogens Fusarium oxysporum f. sp. cucumerinum, and Fusarium oxysporium f. sp. Niveum, but the former pathogen was more sensitive to the inhibitory effects of those exudates than the latter one (Zhou et al., 2011). Yuan and Wu (2006) reported that wheat, mung bean and radish were more sensitive to persimmon leaf extracts than tomato, Chinese cabbage and cucumber. Additionally, Dorning and Cipollini (2006) explored the potential of aqueous extracts of the leaves and roots of Lonicera maackii for inhibiting seed germination of its own species and of three herbs in petri dish bioassays. The results indicated that L. maackii root and leaf extracts significantly decreased germination in the three herb species, and the inhibitory effect generally increased with increasing extract concentration. However, the same extracts and concentration did not decrease seed germination of L. maackii itself, and some of the extract treatments even significantly increased the seed germination of its own species when compared to no-extract controls (Dorning & Cipollini, 2006). Wang et al. (2011) reported that the allelopathic succession effects of Artemisia sacrorum on the seed germination and seedling growth of four dominant or accompanying species differed with varying donor plant extract concentrations and with varying receptors. Moreover, Lei et al. (2010) reported that the allelopathic effects of ginseng root exudates on the seed germination of four medicinal plants were concentration and receptor dependent.

Allelopathy also has selectivity for growth indices of the same receptor. Zhang et al. (2010)) found that an aqueous extract of Allium macrostem, when at the same concentration, caused more inhibition on root length than on shoot length of three weeds, Digitaria sanguinalis, Echinochloa crusgalli, and Amaranthus retroflexus. Jiang et al. (2006) reported that the water extract of Lycoris radiate inhibited the shoots more than the roots of cucumber, rape, and radish. Ma et al. (2011) tested the allelopathy of root exudates of 40 wheat cultivars on cucumber by bioassay and found differences in the allelopathy between the same wheat cultivar on different growth indices of cucumber. Zhang et al. (2011) tested the allelopathy of ginseng root exudates on seed germination and biomass of its own species and American ginseng and observed that those exudates exhibited a stronger allelopathic effect on its own species than on American ginseng. Furthermore, those inhibitory effects were concentration dependent and varied with the hypocotyl growth of both plant types.

In our research, lily root exudates that were collected at the same stage of hydroponic culture showed different allelopathic effects on the four studied receptors. Radish was found to be the most sensitive and lettuce was the least sensitive receptor to lily root exudates. Additionally, lily root exudates had different allelopathic effects on different growth indices of the same receptor. For example, the root length of radish was more sensitive than other indices to lily root exudates that were collected after 10 days of hydroponic culture, but the root fresh weight of lettuce, tomato, and cucumber was more sensitive to lily root exudates than other indices. These results are in agreement with the results of Ma et al. (2011) and Zhang et al. (2011). Furthermore, radish seems to be the most rational receptor for bioassays of the allelopathy of lily root exudates, while lettuce and cucumber are the most suitable after-crops for lily.

The allelopathy intricacy of lily root exudates that were collected at different hydroponic culture stages

Commonly, the allelopathy of plant root exudates strengthens during plant growth because of increased viscosity and the continuous accumulation of allelochemicals. Allelopathy may be different as a result of differences in the concentration of allelochemicals that accumulate at different growth stages of the plant (Yu et al., 2003; Geng et al., 2009; Guo et al., 2010). Some differences in the allelopathy of lily root exudates that were collected at different stages of hydroponic culture were seen in this research. As shown in Figure 1, lily root exudates that were collected after 10 days of hydroponic culture presented a positive effect on the most sensitive receptor, radish, but the exudates that were collected after 20 days and 30 days of hydroponic culture exhibited inhibitory effects on this receptor. These results demonstrate that lily root exudates have a positive effect at lower concentrations and play an inhibitory role when at higher concentrations. However, on less sensitive receptors, i.e., tomato and cucumber, and the least sensitive receptor, i.e., lettuce, the allelopathy of lily root exudates did not always follow the concentration effect rule (i.e., promotion at lower concentrations and inhibition at higher concentrations). For example, lily root exudates that were collected after 20 days of hydroponic culture showed the weakest allelopathic effect on tomato and cucumber and the strongest allelopathic effect on lettuce when compared to exudates that were collected after 10 and 30 days of hydroponic culture. These results demonstrate the allelopathic intricacy of lily root exudates. In addition to the concentration effect rule, a difference in allelochemicals from root exudates that were collected at different hydroponic culture or plant growth stages was observed. That is, at different growth stages, different types of allelochemicals could be produced, a change in the ratio of the constituents could occur or these factors could coexist. Furthermore, the allelopathy compound effect might also contribute to the difference in allelopathy. The compound effect normally appears in three forms: cooperative, additive, and antagonistic (Blum, 1996). However, the effect caused by a micro-organism that is present at different growth stages cannot be neglected without aseptic conditions. To find the definite reasons for the complex allelopathic effects of lily root exudates, further investigations are suggested.

Collection method of lily root exudates

Many methods exist for collecting plant root exudates such as soil, sand, and hydroponic culture collections or a successive root exudate collection system. Each system has its own benefits and limitations. Soil and sand cultures are natural but complex processes and are easily interfered with by soil substances and microorganisms (Tu & Wu, 2010). Hydroponic culture is simple and reliable but sometimes complex because of desalting and infection by microorganisms (Tu & Wu, 2010). The successive root exudate collection system is a good method to detect the allelopathy of plant root exudates, but it requires complex equipment and is not applicable to large-scale research (Tu & Wu, 2010). In our research, lily root exudates were collected by hydroponic culture, and a nutrient solution was substituted by water because enough nutrients were stored in lily bulbs to ensure normal root and shoot growth. This method is convenient and proved reliable in garlic root exudate extraction (Khan et al., 2011).

Through the exudation of a wide variety of compounds, roots impact the microbial community of the soil in their immediate vicinity, influence the resistance of plants to pests, support beneficial symbioses, alter the chemical and physical properties of the soil, and affect the growth of itself, its neighbors and after-crops (Bertin et al., 2003). These functions are dependent on the composition and concentration of root exudates. Therefore, it is suggested that the allelochemical composition of lily root exudates be identified in subsequent studies. Furthermore, changes in the composition and concentration of allelochemicals may also be investigated at varying growth stages.

Acknowledgements

This research was supported by the State Natural Science Foundation (No.31171949) and the National Key Technologies R&D Program of China during the 11th five-year plan period (No.2006BAD07B02).