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Abstract Black pine （Pinus thunbergii） is one of the main afforestation species in the sandy coast and the preferred tree species for landscape green in northern China. Allelopathic activity could be one of the reasons of the sparse vegetation under black pine forests. To explore the allelopathic potential of P. thunbergii， the effects of litter and fresh needles were evaluated against the common cover plants seeds and seedlings， i.e. Tateges erecta， Orychophragmus violaceus， Ophiopogon japonicas and Trifolium repens. The results showed that P. thunbergii possessed allelopathic potential to the accompanying plants. Increasing concentrations of aqueous extracts of needles and litter affected target plant development in different levels. Growth and germination inhibition were directly related to the extract concentrations of needles or litters. Fresh needles played a more important role in the inhibition to target plants than litter because of the less germination rate and biomass in the same concentration. In addition， the adaptability of cover plants to the allelopathic effects of P. thunbergii is obviously different. The sensitivities of the four cover plants to the allelopathic effects of P. thunbergii were in order of T. repens>O. japonicas>O. violaceus> T. erecta. Therefore， T. repens is least suitable for concomitant planting with P. thunbergii， and T. erecta is the most suitable， with other two plants in between in landscape configuration.
Key words Pinus thunbergii; Allelopathy; Litter; Cover plants
Black pine （Pinus thunbergii） has the characteristics of antisea breeze， barren and adaptability. In the neutral or slightly alkaline sand beach， P. thunbergii can grow well and is one of the main afforestation species in the sandy coast in northern China. At present， P. thunbergii has also become the preferred tree species for landscape green. However， the black pine forest vegetation is relatively simple： shrubs mainly include Amorpha fruticosa， Vitex， Lespedeza， wild roses and other trees， and the herb layer mainly have Eriophorum， Portulaca oleracea， Calystegia soldanella， Ischaemum indicum， Artemisia capillaries Thunb， Commelina communis and Solanum nigrum and so on， mostly in sporadic distribution. Compared with the shrub community on the edge of the P. thunbergii forest， the species diversity is significantly reduced[1]. The sparse vegetation under black pine cultivated forests could be the result of allelopathic activity， besides physical limitations， such as shading and heavy litter accumulation[2]. Allelopathy means that plants promote or inhibit the growth of other plants in the microenvironment area by releasing some organic chemical substances into the environment and forming a microenvironment area around the plant[3]. It was reported that several pine species showed strong allelopathic potential[3-5]. The vegetation under red pine forests is sparse compared with other forests that have dense undergrowths of herbaceous plants， despite the fact that the sunlight intensity under red pine forests is sufficient for these herbaceous plants to grow[3].
Studying whether the allelopathic effect and intensity of P. thunbergii on common cover plants is not only helpful to guide the application of P. thunbergii in landscape green， but also important for building a stable garden plant community and ecosystem. The studies are lacking， particularly considering the variables of fresh and litter needles of P. thunbergi. It has not yet been explained whether P. thunbergii will have an allelopathic inhibitory effect on understory species. The object of this work was to verify the allelopathic activity of P. thunbergii on cover plants based on the analysis of the seed germination and seedling growth and identify its ecological significance and application potential in landscape configuration.
Materials and Methods
Experimetal materials
Fresh （green） needles and litter （surface layer） were collected from a black pine plantation （near Huaian city， Jiangsu Province， China; 33°36′41″N， 119°0′37″E） and tested against the seedlings and seeds of four cover plant， i.e. Tateges erecta （marigold）， Orychophragmus violaceus （february orchid）， Ophiopogon japonicas （ liriope） and Trifolium repens （white clover）.
Preparation of extracts
The different plant materials， fresh pine needles and litter （Equivalent to the amount of fresh pine needles by measuring the moisture content）， respectively， were macerated with 500 ml of boiling distilled water （at 100 ℃ at the beginning of the extraction， then later kept at 30 ℃） for 48 h. Increasing concentrations （2%， 4%， 8%， and 16% dw/v） of hot aqueous extracts were prepared with needles or litter collected. After this period， the extracts were filtered and immediately used in the bioassays.
Germination assays
The seeds of marigdd， february orchid， liriope and white clover were sterilized with 0.5% potassium permanganate solution soaking for about 10 min， then rinsed with distilled water for 2-3 times， and dried under natural conditions， respectively. One hundred fifty seeds， were distributed in petri dishes （50 seedlings per dish） containing 5 ml of distilled water （control） or increasing concentrations of black pine aqueous extracts. Petri dishes were sealed with plastic film and kept under controlled light （50 μmol photons/m 2·s） and temperature （25 ℃） for 7 d. The radicle emergence was the germination criterion evaluated. At the end of the 7th d， the germination rate was counted， and the length of hypocotyls and radicles of sprouting seeds was measured. Potting assays
Soil was collected from the surface soil near the black pine plantation. The soil was loaded into the pots （volume 4 L）. The extract concentrate of needles or litter was mixed in the ratio of 0， 2%， 4%， 8% and 16%， （dw/v）， respectively. White clover with 4 leaves was transplanted and cultivated for 60 d， 15 plants per pot. All pots were watered by distilled water once every 3 d. Three replicates of 15 pots each were evaluated. At the end of this period， the biomass of the plant was measured after drying at 65 ℃.
Statistical analysis
Statistical analyses were carried out using SPSS 17.0 and Excel 2003 for Windows. All of the results were reported as the means ± standard error （SE） for three replicates. The means were separated by the leastsignificant difference （LSD） test， with a significance level of P<0.05 throughout the study.
Results and Discussion
Effects of black pine needles and litter on seed germination of four cover plants
Various concentrations of aqueous extracts of black pine affected germination of the four cover plants. In general， needles extracts inhibited the seed germination and had obvious effect on seed germination rate. By contrast， the effect of fresh needle on seed germination was more significant than litter because of the less germination rate in the same concentration. For fresh needles， the germination rate decreased obviously with the increase of concentration of aqueous extracts. The seed germination rate of marigold， february orchid， liriope and white clover decreased by 24.2%， 65.9%， 67.0% and 87.3% in 8% extract concentration， respectively， and no seed germinated in 16% extract concentration for the three plants except for marigold. For litter， increasing concentrations of aqueous extracts also inhibited seed germination. But compared with fresh needles， the rate decline slowed down and the germination rate increased at some degree. In 8% extract concentration， the seed germination rate of the four plants above decreased by 9.2%， 37.0%， 41.9% and 79.7%， respectively. Only white clover still had no germination in 16% extract concentration， while others had a certain germination rate （Table 1）.
Effects of black pine needles and litter on seedling growth of four cover plants
Various concentrations of aqueous extracts of black pine affected early growth of the four cover plants in different levels. More and more significant inhibitory effect on cover plants growth was observed in the radicles and hypocotyls length with increase of extract concentration. In higher extract concentrations （8% and 16%）， the effect of fresh needle extracts was more conspicuous compared to litter （Tables 2 and 3）. Among the four plants， marigold was the least affected and clover was the most affected by pine extract. Firstly， among the four cover plants， it was most significant for the germination and growth of white clover inhibited by the extract of black pine. Particularly， no seed germination and seedling growth were observed in the highest extract concentration prepared with fresh needles or litter （Tables 1， 2 and 3）. Secondly， it was interesting that hypocotyl of liriope was not found regardless of seed germination and radicle growth observed in 16% litter concentration. It may mean that the litter extract weakened further the later seedling growth of liriope based on the early effect. The hypocotyls and radicle growth of february orchid was affected in similar fashion， and the inhibition effect was directly related to the extract concentrations. These data showed that seedling growth was inhibited obviously in the 2% concentration of fresh needles， whereas the higher （4%） concentration of litter. In the 16% litter concentration， its radicle and hypocotyl length decreased by 71.7% and 27.3% compared with the control， respectively. In addition， the pine extract of higher concentration （8% and 16%） showed significant inhibitory or reducing effects to the radicle and hypocotyl length of marigold， those of lower ones showed no effects （Tables 2 and 3）.
The results above suggest that the pine needles possess allelopathic potential and the extract of pine needles contain growth inhibitory substances. And fresh needles may play a more important role in the allelopathy to cover plants than litter. Allelopathic effects of aqueous extract of black pine on the cover plants were significantly different. Overall， white clover is the most sensitive receptor plant to the allelopathy of black pine， compared with the others. Marigold showed the best adaptability to the allelopathy.
Agricultural Biotechnology2018
Effects of black pine needles on plant biomass of white clover
It can be seen from the seed germination and seedling growth of plants that white clover was the most sensitive receptor plant to the allelopathy of black pine compared to the other three cover plants （Tables 1， 2 and 3）. White clover was chosen as a representative sensitive plant to study the allelopathy of black pine needles and their litter in soil. The results showed that （Table 4）， low concentrations of fresh pine needles and litter had no significant effect on the growth of white clover， while the addition of high litter significantly inhibited its growth. The soil with 8% and 16% fresh pine needle concentrate had a significant effect on the white clover biomass， and the biomass decreased by 45.9% and 65.0%， respectively. Although the soil with the addition of litter concentrate also had a significant effect on the white clover， its impact was relatively smaller， and the biomass decreased by 17.1% and 35.7% under the condition of 8% and 16% addition， respectively. Allelopathic effects of plant allelochemicals on recipient plants are closely related to plant species and their concentrations[6]. The allelopathic effects of soil leachates， green needles and litter extracts of Pinus spp. on germination and seedling growth aspects of wild and crop species have been evaluated in natural and cultivated pine stands and have proven to be stimulatory or inhibitory[7-17]. Luo et al.[18] indicated that the different organ powders of Pinus， Picea， and Platycladus have an allelopathic activity to the growth of lettuce. Zhang et al.[1] showed that the water extracts from leaf， bark and root of P. thunbergii had the most significant inhibitory effects on the germination rate， radicle length and biomass of Chinese cabbage， respectively. Extracts from P. massoniana rhizosphere soil had obvious allelopathic inhibition effects on the four receptor plants， i.e.， C. dactylon， T. repens， P. notatum and P. annua， under 1.0， 0.5 and 0.25 mg/ml concentrations[19]. In our study， on the one hand， the pine needles and litter water extracts significantly reduced the seed germination and seedling growth of T. erecta， O. violaceus， O. japonicas and T. repens. On the other hand， the pine needle and its litter also had allelopathic effects in the soil， which significantly inhibited the growth of T. repens and reduced its biomass. The result suggests that failure of the cover plants to grow in the pine forest was due to an allelopathic effect of black pine. Allelopathic effects play an important role in resource competition and natural selection of the accompanying plants under natural conditions. Therefore， the release and secretion of organic compounds from P. thunbergii is also a simple cause of vegetation composition. After the leaves and their litters are degraded in the soil， the allelochemicals are released. Plant allelochemicals are mostly plant secondary metabolites[20]. The study found that there was a big difference in the composition （type and content） of the allelopathic components of different tree species[6]. Lin et al.[21] reported that considerable quantities of volatile organic compounds （mainly monoterpenes and sesquiterpenes） were the exudates of rhizosphere of P. pinea and P. sylvestris growing in field conditions. KatoNoguchi et al.[22] showed that 9α， 13 βepidioxyabeit8 （14） en18oic acid was isolated and determined from red pine by spectral data. It suggest that 9α， 13 βepidioxyabeit8 （14） en18oic acid may contribute to the growth inhibitory effect of the pine needles and may play an important role in the allelopathy of red pine. The common organics were dehydroabitetic acid and octadecanoic acid in P. massoniana rhizospheric soil[23]. At present， the main allelochemicals released during the allelopathic process of P. thunbergii and its mechanism on recipient plants remain unclear and need to be further studied， which is the goal of our next study. Overall， the inhibitory effects of aqueous pine needle extracts seem to be directly correlated to extract concentration. The higher the concentration of the water extract was， the more obvious effect it had. Even no seed germinated in 16% extract concentration. Similar results had been observed in other Pinus spp. The highest concentration of P. pinea L. （stone pine） aqueous extract tested against C. salvifolius， C. libanotis and H. halimifolium presented the most deleterious effects on seedling growth and seed germination[16].
In addition， RodriguesCorrêa et al.[17] found that litter extracts of P. elliottii promoted the plant growth of L. sativa， whereas needle extracts inhibited growth and delayed germination. That was not consistent with the finding of our study. In our study， both of the fresh needles and litters extract existed inhibitory effect to the seeds and seedlings of the four cover plants， but the effect of fresh needle was more significant than litter. It may mean that the time length or the decay degree of litter would affect the allelopathy effect directly. As time goes on， the allelopathic effects may gradually disappear or weaken with further decay of litter. This view would be supported by the following research. Jia et al.[4] found that on the same stand， the effects of higher concentrations of leachates of litter are higher than half decaying litter and surface layer soil on stem growth， the chlorophyll content， the stoma resistance and the net photosynthesis speed of pine seedlings.
Allelopathy is one of the main reasons that affect the success or failure of garden plants and seriously affect the stability and sustainable development of ecosystems. Lee and Monsi[24] suspected that failure of these herbaceous plants to grow in the red pine forest was due to an allelopathic effect of the pines. They investigated the allelopathic potential of the pine against the seed germination of 15 plant species and found that the needle extracts of the pine markedly inhibited the germination of three plant species. Wan et al.[25] found that there existed significant allelopathic effects of A. adenophora on seed germination of Trifolium repens， Galinsoga parviflora and Medicago sativa， which was significantly promoted at low concentration and inhibited at high concentration of the water extracts of A. adenophora leaf litter， while insignificant effect on L. perenne. It is important to explore the allelopathic effect of P. thunbergii on different cover plants for building a stable plant community and ecosystem. And it will be also helpful to guide the application of P. thunbergii in afforestation and landscape configuration. Conclusions
In this study， the allelopathic effects of P. thunbergii aqueous extract on the four recipient plants were significantly different. T. repens showed the strongest sensitivity to allelopathy of P. thunbergii， followed by O. japonicas， O. violaceu and T. erecta. In general， in landscape configuration T. repens is least suitable for concomitant planting with black pine， and T. erecta is the most suitable， with the other two plants in between. Thus， this study provides a new perspective to explain the understory vegetation status by exploring allelopathic effects and intensity of P. thunbergii on understory vegetation. It is helpful to improve the vegetation structure and build a stable plant ecosystem.
References
[1] ZHANG J， WANG GM， QU ZQ， et al. Study on the allelopathy of Pinus thunbergii[J]. Shandong Agricultural Sciences， 2012， 7： 37-40. （In Chinese）
[2] ZHANG ZD， MAO PL， LIU YH， et al. Effects of forest structure on natural regeneration of Pinus thunbergii coastal shelter forest in Yantai region[J]. Acta Ecologica Sinica， 2010， 8： 2205-2211. （In Chinese）
[3] RICE EL. Allelopathy[M]. New York： Academic Press， 1984.
[4] JIA LM， ZHAI MP， FENG CH. Effects of allelopathic substances on the growth and photosynthesis of Pinus tabulaeformis seedlings[J]. Journal of Beijing Forestry University， 2003， 25： 6-10. （In Chinese）
[5] FERNANDEZ C， VOIRIOT S， MéVY JP， et al. Regeneration failure of Pinus halepensis Mill.： The role of autotoxicity and some abiotic environmental parameters[J].Forest Ecology and Management， 2008， 7： 2928-2936.
[6] YANG QH， YE WH， LIAO FL， et al. Effects of allelochemicals on seed germination[J]. Chinese Journal of Ecology， 2005， 12： 1459-1465. （In Chinese）
[7] LODHI MAK， KILLINGBECK KT. Effects of pineproduced chemicals on selected understory species in a Pinus ponderosa community[J]. Journal of Chemical Ecology， 1982， 8： 275-283.
[8] KIL BS， YIM YJ. Allelopathic effects of Pinus densiflora on undergrowth of red pine forest[J]. Journal of Chemical Ecology， 1983， 9： 1135-1151.
[9] NEKTARIOS PA， ECONOMOU G， AVGOULAS C. Allelopathic effects of Pinus halepensis needles on turfgrasses and biosensor plants[J]. Hort Science， 2005， 40： 246-250.
[10] AKKAYA A， DOKUYUCU T， KARA R， et al. The effects of walnut and pine leaves on bread wheat growth and frequence of common weed species in the EastMediterranean region[J]. Journal of Environmental Biology， 2006， 27： 523-527. [11] MACHADO S. Allelopathic potential of various plant species on downy brome： implications for weed control in wheat production[J]. Agron J， 2007， 99： 127-132.
[12] ALRABABAH MA， TADROS MJ， SAMARAH NH， et al. Allelopathic effects of Pinus halepensis and Quercus coccifera on the germination of Mediterranean crop seeds[J]. New Forests， 2009， 38： 261-272.
[13] SARTOR LR， ADAMI PF， CHINI N， et al. Allelopathy of Pinus taeda needles on the germination and development of Avena strigosa seedlings[J]. Ciência Rural， 2009， 39： 1653-1659.
[14] KATONOGUCHI H， FUSHIMI Y， SHIGEMORI H. An allelopathic substance in red pine needles （Pinus densiflora）[J]. Journal of Plant Physiology， 2009， 166： 442-446.
[15] ROLON AS， ROCHA O， MALTCHIK L. Does pine occurrence influence the macrophyte assemblage in Southern Brazil ponds[J]. Hydrobiologia， 2011， 675： 157-165.
[16] VALERABURGOS J， DíAZBARRADAS MC， ZUNZUNEGUI M. Effects of Pinus pinea litter on seed germination and seedling performance of four Mediterranean shrub species[J]. Plant Growth Regulation， 2012， 66： 285-292.
[17] RODRIGUESCORRA KCS， HALMENSCHLAGER G， SCHWAMBACH J， et al. Dual allelopathic effects of subtropical slash pine （Pinus elliottii Engelm.） needles： Leads for using a large biomass reservoir[J]. Industrial Crops & Products， 2017， 108： 113-120.
[18] LUO XY， REN YX， ZHOU SJ. Allelopathic activities of some Pinaceae， Cupressaceae and Taxodiaceae plants[J]. Weed Science， 2008， 4： 22-25. （In Chinese）
[19] DUAN J， WANG LY， YANG J， et al. Chemical constituents in rhizospheric soil extracts of Pinus massoniana and Liquidambar formosana[J]. Scientia Silvae Sinicae， 2015， 8： 8-15. （In Chinese）
[20] MINORSKY PV. Different roles for Catechin enantiomers secreted into rhizosphere[J]. Plant Physiology， 2002， 128（4）： 1163-1164.
[21] LIN C， OWEN SM， PEЙUELAS J. Volatile organic compounds in the roots and rhizosphere of Pinus spp. [J]. Soil Biology & Biochemistry， 2007， 39： 951-960. （In Chinese）
[22] KATONOGUCHI H， FUSHIMI Y， TANAKA Y， et al. Allelopathy of red pine： isolation and identification of an allelopathic substance in red pine needles[J]. Plant Growth Regulation， 2011， 65： 299-304.
[23] DUAN J， YANG J， KANG JL， et al. Allelopathic effect of extracts from Pinus massoniana rhizosphere soil[J]. Chinese Journal of Ecology， 2015， 3： 773-780. （In Chinese）
[24] LEE IK， MONSI M. Ecological studies on Pinus densiflora forest I. Effects of plant substances on the floristic composition of the undergrowth[J]. Botanical Magazyne， 1963， 76： 400-413.
[25] WAN HH， LIU WX， WAN FH. Allelopathic effect of Ageratina adenophora （Spreng.） leaf litter on four herbaceous plants in invaded regions[J]. Chinese Journal of EcoAgriculture， 2011， 1： 130-134. （In Chinese）.
Key words Pinus thunbergii; Allelopathy; Litter; Cover plants
Black pine （Pinus thunbergii） has the characteristics of antisea breeze， barren and adaptability. In the neutral or slightly alkaline sand beach， P. thunbergii can grow well and is one of the main afforestation species in the sandy coast in northern China. At present， P. thunbergii has also become the preferred tree species for landscape green. However， the black pine forest vegetation is relatively simple： shrubs mainly include Amorpha fruticosa， Vitex， Lespedeza， wild roses and other trees， and the herb layer mainly have Eriophorum， Portulaca oleracea， Calystegia soldanella， Ischaemum indicum， Artemisia capillaries Thunb， Commelina communis and Solanum nigrum and so on， mostly in sporadic distribution. Compared with the shrub community on the edge of the P. thunbergii forest， the species diversity is significantly reduced[1]. The sparse vegetation under black pine cultivated forests could be the result of allelopathic activity， besides physical limitations， such as shading and heavy litter accumulation[2]. Allelopathy means that plants promote or inhibit the growth of other plants in the microenvironment area by releasing some organic chemical substances into the environment and forming a microenvironment area around the plant[3]. It was reported that several pine species showed strong allelopathic potential[3-5]. The vegetation under red pine forests is sparse compared with other forests that have dense undergrowths of herbaceous plants， despite the fact that the sunlight intensity under red pine forests is sufficient for these herbaceous plants to grow[3].
Studying whether the allelopathic effect and intensity of P. thunbergii on common cover plants is not only helpful to guide the application of P. thunbergii in landscape green， but also important for building a stable garden plant community and ecosystem. The studies are lacking， particularly considering the variables of fresh and litter needles of P. thunbergi. It has not yet been explained whether P. thunbergii will have an allelopathic inhibitory effect on understory species. The object of this work was to verify the allelopathic activity of P. thunbergii on cover plants based on the analysis of the seed germination and seedling growth and identify its ecological significance and application potential in landscape configuration.
Materials and Methods
Experimetal materials
Fresh （green） needles and litter （surface layer） were collected from a black pine plantation （near Huaian city， Jiangsu Province， China; 33°36′41″N， 119°0′37″E） and tested against the seedlings and seeds of four cover plant， i.e. Tateges erecta （marigold）， Orychophragmus violaceus （february orchid）， Ophiopogon japonicas （ liriope） and Trifolium repens （white clover）.
Preparation of extracts
The different plant materials， fresh pine needles and litter （Equivalent to the amount of fresh pine needles by measuring the moisture content）， respectively， were macerated with 500 ml of boiling distilled water （at 100 ℃ at the beginning of the extraction， then later kept at 30 ℃） for 48 h. Increasing concentrations （2%， 4%， 8%， and 16% dw/v） of hot aqueous extracts were prepared with needles or litter collected. After this period， the extracts were filtered and immediately used in the bioassays.
Germination assays
The seeds of marigdd， february orchid， liriope and white clover were sterilized with 0.5% potassium permanganate solution soaking for about 10 min， then rinsed with distilled water for 2-3 times， and dried under natural conditions， respectively. One hundred fifty seeds， were distributed in petri dishes （50 seedlings per dish） containing 5 ml of distilled water （control） or increasing concentrations of black pine aqueous extracts. Petri dishes were sealed with plastic film and kept under controlled light （50 μmol photons/m 2·s） and temperature （25 ℃） for 7 d. The radicle emergence was the germination criterion evaluated. At the end of the 7th d， the germination rate was counted， and the length of hypocotyls and radicles of sprouting seeds was measured. Potting assays
Soil was collected from the surface soil near the black pine plantation. The soil was loaded into the pots （volume 4 L）. The extract concentrate of needles or litter was mixed in the ratio of 0， 2%， 4%， 8% and 16%， （dw/v）， respectively. White clover with 4 leaves was transplanted and cultivated for 60 d， 15 plants per pot. All pots were watered by distilled water once every 3 d. Three replicates of 15 pots each were evaluated. At the end of this period， the biomass of the plant was measured after drying at 65 ℃.
Statistical analysis
Statistical analyses were carried out using SPSS 17.0 and Excel 2003 for Windows. All of the results were reported as the means ± standard error （SE） for three replicates. The means were separated by the leastsignificant difference （LSD） test， with a significance level of P<0.05 throughout the study.
Results and Discussion
Effects of black pine needles and litter on seed germination of four cover plants
Various concentrations of aqueous extracts of black pine affected germination of the four cover plants. In general， needles extracts inhibited the seed germination and had obvious effect on seed germination rate. By contrast， the effect of fresh needle on seed germination was more significant than litter because of the less germination rate in the same concentration. For fresh needles， the germination rate decreased obviously with the increase of concentration of aqueous extracts. The seed germination rate of marigold， february orchid， liriope and white clover decreased by 24.2%， 65.9%， 67.0% and 87.3% in 8% extract concentration， respectively， and no seed germinated in 16% extract concentration for the three plants except for marigold. For litter， increasing concentrations of aqueous extracts also inhibited seed germination. But compared with fresh needles， the rate decline slowed down and the germination rate increased at some degree. In 8% extract concentration， the seed germination rate of the four plants above decreased by 9.2%， 37.0%， 41.9% and 79.7%， respectively. Only white clover still had no germination in 16% extract concentration， while others had a certain germination rate （Table 1）.
Effects of black pine needles and litter on seedling growth of four cover plants
Various concentrations of aqueous extracts of black pine affected early growth of the four cover plants in different levels. More and more significant inhibitory effect on cover plants growth was observed in the radicles and hypocotyls length with increase of extract concentration. In higher extract concentrations （8% and 16%）， the effect of fresh needle extracts was more conspicuous compared to litter （Tables 2 and 3）. Among the four plants， marigold was the least affected and clover was the most affected by pine extract. Firstly， among the four cover plants， it was most significant for the germination and growth of white clover inhibited by the extract of black pine. Particularly， no seed germination and seedling growth were observed in the highest extract concentration prepared with fresh needles or litter （Tables 1， 2 and 3）. Secondly， it was interesting that hypocotyl of liriope was not found regardless of seed germination and radicle growth observed in 16% litter concentration. It may mean that the litter extract weakened further the later seedling growth of liriope based on the early effect. The hypocotyls and radicle growth of february orchid was affected in similar fashion， and the inhibition effect was directly related to the extract concentrations. These data showed that seedling growth was inhibited obviously in the 2% concentration of fresh needles， whereas the higher （4%） concentration of litter. In the 16% litter concentration， its radicle and hypocotyl length decreased by 71.7% and 27.3% compared with the control， respectively. In addition， the pine extract of higher concentration （8% and 16%） showed significant inhibitory or reducing effects to the radicle and hypocotyl length of marigold， those of lower ones showed no effects （Tables 2 and 3）.
The results above suggest that the pine needles possess allelopathic potential and the extract of pine needles contain growth inhibitory substances. And fresh needles may play a more important role in the allelopathy to cover plants than litter. Allelopathic effects of aqueous extract of black pine on the cover plants were significantly different. Overall， white clover is the most sensitive receptor plant to the allelopathy of black pine， compared with the others. Marigold showed the best adaptability to the allelopathy.
Agricultural Biotechnology2018
Effects of black pine needles on plant biomass of white clover
It can be seen from the seed germination and seedling growth of plants that white clover was the most sensitive receptor plant to the allelopathy of black pine compared to the other three cover plants （Tables 1， 2 and 3）. White clover was chosen as a representative sensitive plant to study the allelopathy of black pine needles and their litter in soil. The results showed that （Table 4）， low concentrations of fresh pine needles and litter had no significant effect on the growth of white clover， while the addition of high litter significantly inhibited its growth. The soil with 8% and 16% fresh pine needle concentrate had a significant effect on the white clover biomass， and the biomass decreased by 45.9% and 65.0%， respectively. Although the soil with the addition of litter concentrate also had a significant effect on the white clover， its impact was relatively smaller， and the biomass decreased by 17.1% and 35.7% under the condition of 8% and 16% addition， respectively. Allelopathic effects of plant allelochemicals on recipient plants are closely related to plant species and their concentrations[6]. The allelopathic effects of soil leachates， green needles and litter extracts of Pinus spp. on germination and seedling growth aspects of wild and crop species have been evaluated in natural and cultivated pine stands and have proven to be stimulatory or inhibitory[7-17]. Luo et al.[18] indicated that the different organ powders of Pinus， Picea， and Platycladus have an allelopathic activity to the growth of lettuce. Zhang et al.[1] showed that the water extracts from leaf， bark and root of P. thunbergii had the most significant inhibitory effects on the germination rate， radicle length and biomass of Chinese cabbage， respectively. Extracts from P. massoniana rhizosphere soil had obvious allelopathic inhibition effects on the four receptor plants， i.e.， C. dactylon， T. repens， P. notatum and P. annua， under 1.0， 0.5 and 0.25 mg/ml concentrations[19]. In our study， on the one hand， the pine needles and litter water extracts significantly reduced the seed germination and seedling growth of T. erecta， O. violaceus， O. japonicas and T. repens. On the other hand， the pine needle and its litter also had allelopathic effects in the soil， which significantly inhibited the growth of T. repens and reduced its biomass. The result suggests that failure of the cover plants to grow in the pine forest was due to an allelopathic effect of black pine. Allelopathic effects play an important role in resource competition and natural selection of the accompanying plants under natural conditions. Therefore， the release and secretion of organic compounds from P. thunbergii is also a simple cause of vegetation composition. After the leaves and their litters are degraded in the soil， the allelochemicals are released. Plant allelochemicals are mostly plant secondary metabolites[20]. The study found that there was a big difference in the composition （type and content） of the allelopathic components of different tree species[6]. Lin et al.[21] reported that considerable quantities of volatile organic compounds （mainly monoterpenes and sesquiterpenes） were the exudates of rhizosphere of P. pinea and P. sylvestris growing in field conditions. KatoNoguchi et al.[22] showed that 9α， 13 βepidioxyabeit8 （14） en18oic acid was isolated and determined from red pine by spectral data. It suggest that 9α， 13 βepidioxyabeit8 （14） en18oic acid may contribute to the growth inhibitory effect of the pine needles and may play an important role in the allelopathy of red pine. The common organics were dehydroabitetic acid and octadecanoic acid in P. massoniana rhizospheric soil[23]. At present， the main allelochemicals released during the allelopathic process of P. thunbergii and its mechanism on recipient plants remain unclear and need to be further studied， which is the goal of our next study. Overall， the inhibitory effects of aqueous pine needle extracts seem to be directly correlated to extract concentration. The higher the concentration of the water extract was， the more obvious effect it had. Even no seed germinated in 16% extract concentration. Similar results had been observed in other Pinus spp. The highest concentration of P. pinea L. （stone pine） aqueous extract tested against C. salvifolius， C. libanotis and H. halimifolium presented the most deleterious effects on seedling growth and seed germination[16].
In addition， RodriguesCorrêa et al.[17] found that litter extracts of P. elliottii promoted the plant growth of L. sativa， whereas needle extracts inhibited growth and delayed germination. That was not consistent with the finding of our study. In our study， both of the fresh needles and litters extract existed inhibitory effect to the seeds and seedlings of the four cover plants， but the effect of fresh needle was more significant than litter. It may mean that the time length or the decay degree of litter would affect the allelopathy effect directly. As time goes on， the allelopathic effects may gradually disappear or weaken with further decay of litter. This view would be supported by the following research. Jia et al.[4] found that on the same stand， the effects of higher concentrations of leachates of litter are higher than half decaying litter and surface layer soil on stem growth， the chlorophyll content， the stoma resistance and the net photosynthesis speed of pine seedlings.
Allelopathy is one of the main reasons that affect the success or failure of garden plants and seriously affect the stability and sustainable development of ecosystems. Lee and Monsi[24] suspected that failure of these herbaceous plants to grow in the red pine forest was due to an allelopathic effect of the pines. They investigated the allelopathic potential of the pine against the seed germination of 15 plant species and found that the needle extracts of the pine markedly inhibited the germination of three plant species. Wan et al.[25] found that there existed significant allelopathic effects of A. adenophora on seed germination of Trifolium repens， Galinsoga parviflora and Medicago sativa， which was significantly promoted at low concentration and inhibited at high concentration of the water extracts of A. adenophora leaf litter， while insignificant effect on L. perenne. It is important to explore the allelopathic effect of P. thunbergii on different cover plants for building a stable plant community and ecosystem. And it will be also helpful to guide the application of P. thunbergii in afforestation and landscape configuration. Conclusions
In this study， the allelopathic effects of P. thunbergii aqueous extract on the four recipient plants were significantly different. T. repens showed the strongest sensitivity to allelopathy of P. thunbergii， followed by O. japonicas， O. violaceu and T. erecta. In general， in landscape configuration T. repens is least suitable for concomitant planting with black pine， and T. erecta is the most suitable， with the other two plants in between. Thus， this study provides a new perspective to explain the understory vegetation status by exploring allelopathic effects and intensity of P. thunbergii on understory vegetation. It is helpful to improve the vegetation structure and build a stable plant ecosystem.
References
[1] ZHANG J， WANG GM， QU ZQ， et al. Study on the allelopathy of Pinus thunbergii[J]. Shandong Agricultural Sciences， 2012， 7： 37-40. （In Chinese）
[2] ZHANG ZD， MAO PL， LIU YH， et al. Effects of forest structure on natural regeneration of Pinus thunbergii coastal shelter forest in Yantai region[J]. Acta Ecologica Sinica， 2010， 8： 2205-2211. （In Chinese）
[3] RICE EL. Allelopathy[M]. New York： Academic Press， 1984.
[4] JIA LM， ZHAI MP， FENG CH. Effects of allelopathic substances on the growth and photosynthesis of Pinus tabulaeformis seedlings[J]. Journal of Beijing Forestry University， 2003， 25： 6-10. （In Chinese）
[5] FERNANDEZ C， VOIRIOT S， MéVY JP， et al. Regeneration failure of Pinus halepensis Mill.： The role of autotoxicity and some abiotic environmental parameters[J].Forest Ecology and Management， 2008， 7： 2928-2936.
[6] YANG QH， YE WH， LIAO FL， et al. Effects of allelochemicals on seed germination[J]. Chinese Journal of Ecology， 2005， 12： 1459-1465. （In Chinese）
[7] LODHI MAK， KILLINGBECK KT. Effects of pineproduced chemicals on selected understory species in a Pinus ponderosa community[J]. Journal of Chemical Ecology， 1982， 8： 275-283.
[8] KIL BS， YIM YJ. Allelopathic effects of Pinus densiflora on undergrowth of red pine forest[J]. Journal of Chemical Ecology， 1983， 9： 1135-1151.
[9] NEKTARIOS PA， ECONOMOU G， AVGOULAS C. Allelopathic effects of Pinus halepensis needles on turfgrasses and biosensor plants[J]. Hort Science， 2005， 40： 246-250.
[10] AKKAYA A， DOKUYUCU T， KARA R， et al. The effects of walnut and pine leaves on bread wheat growth and frequence of common weed species in the EastMediterranean region[J]. Journal of Environmental Biology， 2006， 27： 523-527. [11] MACHADO S. Allelopathic potential of various plant species on downy brome： implications for weed control in wheat production[J]. Agron J， 2007， 99： 127-132.
[12] ALRABABAH MA， TADROS MJ， SAMARAH NH， et al. Allelopathic effects of Pinus halepensis and Quercus coccifera on the germination of Mediterranean crop seeds[J]. New Forests， 2009， 38： 261-272.
[13] SARTOR LR， ADAMI PF， CHINI N， et al. Allelopathy of Pinus taeda needles on the germination and development of Avena strigosa seedlings[J]. Ciência Rural， 2009， 39： 1653-1659.
[14] KATONOGUCHI H， FUSHIMI Y， SHIGEMORI H. An allelopathic substance in red pine needles （Pinus densiflora）[J]. Journal of Plant Physiology， 2009， 166： 442-446.
[15] ROLON AS， ROCHA O， MALTCHIK L. Does pine occurrence influence the macrophyte assemblage in Southern Brazil ponds[J]. Hydrobiologia， 2011， 675： 157-165.
[16] VALERABURGOS J， DíAZBARRADAS MC， ZUNZUNEGUI M. Effects of Pinus pinea litter on seed germination and seedling performance of four Mediterranean shrub species[J]. Plant Growth Regulation， 2012， 66： 285-292.
[17] RODRIGUESCORRA KCS， HALMENSCHLAGER G， SCHWAMBACH J， et al. Dual allelopathic effects of subtropical slash pine （Pinus elliottii Engelm.） needles： Leads for using a large biomass reservoir[J]. Industrial Crops & Products， 2017， 108： 113-120.
[18] LUO XY， REN YX， ZHOU SJ. Allelopathic activities of some Pinaceae， Cupressaceae and Taxodiaceae plants[J]. Weed Science， 2008， 4： 22-25. （In Chinese）
[19] DUAN J， WANG LY， YANG J， et al. Chemical constituents in rhizospheric soil extracts of Pinus massoniana and Liquidambar formosana[J]. Scientia Silvae Sinicae， 2015， 8： 8-15. （In Chinese）
[20] MINORSKY PV. Different roles for Catechin enantiomers secreted into rhizosphere[J]. Plant Physiology， 2002， 128（4）： 1163-1164.
[21] LIN C， OWEN SM， PEЙUELAS J. Volatile organic compounds in the roots and rhizosphere of Pinus spp. [J]. Soil Biology & Biochemistry， 2007， 39： 951-960. （In Chinese）
[22] KATONOGUCHI H， FUSHIMI Y， TANAKA Y， et al. Allelopathy of red pine： isolation and identification of an allelopathic substance in red pine needles[J]. Plant Growth Regulation， 2011， 65： 299-304.
[23] DUAN J， YANG J， KANG JL， et al. Allelopathic effect of extracts from Pinus massoniana rhizosphere soil[J]. Chinese Journal of Ecology， 2015， 3： 773-780. （In Chinese）
[24] LEE IK， MONSI M. Ecological studies on Pinus densiflora forest I. Effects of plant substances on the floristic composition of the undergrowth[J]. Botanical Magazyne， 1963， 76： 400-413.
[25] WAN HH， LIU WX， WAN FH. Allelopathic effect of Ageratina adenophora （Spreng.） leaf litter on four herbaceous plants in invaded regions[J]. Chinese Journal of EcoAgriculture， 2011， 1： 130-134. （In Chinese）.