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Abstract [Objective] The aim was to explain the accumulation characteristic of mineral elements in alpine grassland plants and the effect of supplementary supply on the nutrient changes of mineral elements. [Method] Potted Poa crymophila cv. Qinghai plants were regularly applied with liquid mineral mixed fertilizers， and samples were collected for analysis and detection. [Result] After the regular spraying of liquid mineral mixed fertilizers， the mineral elements in potted P. crymophila and its soil reduced （P
Key words Potted Poa crymophila cv. Qinghai； Mineral element； Accumulation； Liquid mixed fertilizers
Mineral elements are essential nutrients for plant growth and development. Degraded grassland has more accumulation of mineral elements than that of enclosed grassland plants. The Starvation Effect of mineral elements is the endogenetic forces driving the accumulation of grassland mineral elements， and mineral elements are also important responses to grassland succession[1-4]. Is it possible to increase the accumulation of mineral nutrients in alpine grassland plants and soils by spraying liquid mineral mixed fertilizers on a regular basis？ In this paper， potted Poa crymophila cv. Qinghai plants were regularly applied with liquid mineral mixed fertilizers[5]， and samples were collected and analyzed to observe the changes of mineral elements in potted P. crymophila plants and its soil. The aim was to explain the effect of supplementary supply on the nutrient changes of mineral elements in alpine grassland plants. This study was of great significance for the study on the accumulation of mineral elements in degraded grassland plants and its dynamic mechanism， as well as testing the hypothesis of the Starvation Effect of mineral elements. In addition， it could also provide scientific bases and technical support for the restoration and remediation of degraded grassland and the development of ecological livestock husbandry.
Materials and Methods
Test materials
P. crymophila is a species of sparse plexiform grasses with developed roots and stems and strong tillering ability. With upright stems and lush leaves， it can adapt to the complex habitat conditions of the plateau and grow well under different altitude conditions. With the resistances to drought and cold and tolerances to saline??alkaline and barren， it has no strict requirements on the soil. Artificially cultivated P. crymophila grassland can be used as a mowing pasture or served as both mowing and grazing[6]. P. crymophila has soft stems， which taste slightly sweet with good palatability， lush foliage， well??developed vegetative shoots， and a long growing period. After maturity， the branches and leaves remain green and the leaves are not easy to fall off. The crude protein content of leaves in different developmental stages is close to that of alfalfa. Before flowering， the plants are favored by horses， cattle， and sheep. The hay has a soft texture and can be used as a good supplementary feed in winter and spring， and it is also good forage grass for various livestock to gain weights in summers and autumns and to keep the weights in spring and winter[6].
P. crymophila is a kind of grassland plant which is suitable to plant on the Qinghai??Tibet Plateau， and there are commercialized seeds commercially available （the test P. crymophila seeds were provided by De Kejia researcher of the Institute of Grassland of the Academy of Animal Husbandry and Veterinary Science of Qinghai Province）， which are easy to cultivate and have a long growing period. Therefore， P. crymophila is an ideal grassland plant species for grassland plant experiments.
Pot test
The test was conducted in mid??April 2016 in Xining （inside the Northwest Institute of Plateau Biology， Chinese Academy of Sciences）. There were a total of 12 pots of potted P. crymophila （pot diameter of about 30 cm）. The potted soil was grassy black loam （collected from the surrounding pastures around Daban Mountain Haibei Alpine Meadow Localizer Station， mixed with organic fertilizer from cow and sheep manure）， and the seeding rate of P. crymophila was 20 kg/hm2.
Liquid mineral mixed fertilizer： the analytical reagents containing the needed mineral elements were weighed accurately and dissolved in purified water to prepare the liquid mineral mixed fertilizers. The prepared mixed fertilizers contained K of 0.2%， Ca of 0.1%， Mg of 0.04%， Fe of 0.02%， Mn of 0.01%， Zn of 40 mg/L， and Cu of 12 mg/L.
When spraying the liquid mineral fertilizers， the amounts were applied in different gradients， namely， 20 ml/pot， 50 ml/pot and 100 ml/pot， respectively， and the supplement amounts of water were 80 ml/pot， 50 ml/pot and 0 ml/pot， respectively. The control group was irrigated with water of 100 ml/pot. The spraying was done every 10 d from the beginning of May， a total of 10 times. The usual irrigation cycle was 1 time every 1-2 d depending on the weather conditions.
The test lasted until late August， that is， the growth period was 4 months. The above??ground parts of potted P. crymophila were collected according to the test requirements， which were then dried in the shade for later use. Soil samples were prepared by mixing any 3 soils collected randomly from the test pots using a small self??made soil drill， which were also dried in the shade for later use. The collected plant samples were rinsed with deionized water and dried at 60 ??， and the collected soil samples were manually sorted to remove plant roots and other tissues. Then the plant and soil samples were ground and sieved by a 100??mesh sieve. After the pretreatment， the samples were sent to the analysis and testing center of Northwest Institute of Plateau Biology， Chinese Academy of Sciences to perform analysis and testing of various mineral elements.
Analysis test
Analytical instruments： Inductively Coupled Plasma Mass Spectrometer （NexIONTM 350D， PerkinElmer， USA）.
Analysis elements： Ca， Mg， K， Na， P， Cu， Zn， Fe， Mn， and Cr.
Analysis method： standard curve method.
Statistical analysis of data
SPSS20.0 software was used for t test statistical analysis.
Results and Discussion
Mineral elements in potted P. crymophila
The contents of various mineral elements in potted P. crymophila were measured after spraying the self??prepared liquid mineral mixed fertilizers （Table 1 & 2）.
As shown in Table 1 & 2， except Cu， Mn and K， the contents of other mineral elements were reduced to different degrees after spraying the liquid mineral mixed fertilizers. Moreover， the mineral trace elements like Zn， Cr showed significant differences from those of the control （P
In the test， potted P. crymophila plants were more sensitive to the absorption of trace elements than the macroelements， and the trace elements were much easier to accumulate. Compared with the control group， the absorption and accumulation amounts of trace elements in potted P. crymophila were much larger after spraying the liquid mineral mixed fertilizers regularly. For example， the contents of trace elements Cu and Mn in the high fertilization group were increased by 55.2% and 69.2% from the control， respectively， while the contents of the macroelements Ca and P were increased by 4.45% and 5.28%， respectively. According to the hypothesis of the Starvation Effect of mineral elements， the trace elements like Cu， Mn were accumulated in potted P. crymophila because of the insufficient supply during the growth period， and the regular application of liquid mineral mixed fertilizers provided timely supply for most mineral elements in potted P. crymophila， which resulted in no increase of the accumulation. In other words， the accumulation of mineral elements increase due to the lack of timely supply of mineral elements during the growth period. Potted P. crymophila plants were more in need of trace elements during the growing season. In other words， the untimely supply of trace elements in the potted soil environment made the trace elements in potted P. crymophila in the starvation state， in the potting soil environment， and the timely supply of trace elements was more conducive to the growth of potted P. crymophila. There were no P， Cr in the liquid mineral mixed fertilizers. There was no statistically significant difference in the content of P in P. crymophila from the control， while the content of Cr decreased with the increase of spraying amounts， that is， the content of mineral elements in the potted plants of P. crymophila increased due to the application of liquid mineral mixed fertilizers， while there was no significant change in the contents of mineral elements that were not contained in the mixed fertilizers. In other words， the appropriate supply of mineral nutrients could increase the nutrition of the corresponding minerals in the potted plants， which was beneficial to the normal growth of the plants[7-8].
Mineral elements in potted P. crymophila soils
The contents of various mineral elements in potted P. crymophila soils were measured after spraying the self??prepared liquid mineral mixed fertilizers （Table 3 & 4）.
As shown in Table 3 & 4， the contents of mineral elements in the soils were decreased to different degrees after spraying the liquid mineral mixed fertilizers， and showed significant differences from the control （P
On the other hand， the application of liquid mineral mixed fertilizers could also effectively reduce the contents of corresponding mineral elements in potted soils， that is， the application of liquid mineral mixed fertilizers to the potted plants could promote the transportation of more corresponding mineral elements in the soils to the plant tissues （Table 3 & 4）. In other words， compared with the control group， the application of liquid mineral mixed fertilizers could make the potted P. crymophila plants absorb more mineral elements from the soil environment， resulting in the reduction of the corresponding mineral elements in the soils （P
The pot test results showed that the regular supply of liquid mineral elements could reduce the contents of mineral elements in potted plants. According to the Starvation Effect of mineral elements， the regular application （once every 10 d） of liquid mineral mixed fertilizers could make the mineral elements in potted P. crymophila in a state of "non??starvation" due to the timely supply of nutrients， but the potted P. crymophila plants in the control group could not get timely supply， so the mineral elements were accumulated due to the starvation of mineral elements caused by the not timely supply， indicating that the starvation effect was one of the endogenetic forces driving the increase in the accumulation of mineral elements. In the test， the accumulation of mineral elements increased in both the potted plants of P. crymophila and the soils of the control group， while the decrease of mineral elements in both the plants and soils with regular application of the mineral mixed fertilizers （Table 1 & 3， Table 2 & 4）. The supply application of mineral element nutrients could accumulate in the underground root tissues of potted P. crymophila. The results showed that the reduction of mineral elements in potted P. crymophila plants and alpine grassland plants was a timely response and adaptation to the changes in the growth environment， that is， the timely supply of mineral elements could reduce the mineral elements in grassland plants because of the non??starvation state， which was also a phenomenon caused by the Starvation Effect of mineral elements， re??inspecting the hypothesis of the Starvation Effect of mineral elements[1-3].
Conclusion
The regular application of liquid mineral mixed fertilizers can reduce the mineral elements in both the potted P. crymophila plants and soils， suggesting that the supply of mineral elements can effectively increase the applied mineral elements in potted P. crymophila plants， promoting their normal growth. Moreover， it can also promote the transportation of more corresponding mineral elements in the soil to plant issues， thereby reducing the mineral element contents in potted soils.
Compared with the application of liquid mineral mixed fertilizers， the mineral elements are accumulated in the control group since the mineral elements are in a starvation state caused by the untimely supply of mineral elements， indicating that the starvation effect is one of the endogenetic forces driving the increase in the accumulation of mineral elements.References
[1] LI TC. Grassland mineral elements[M]. Beijing： Chemical Industry Press， 2014， 77-83.
[2] LI TC. Study on distribution patterns and accumulation differentiation behavior of mineral elements in grassland on the north bank of Qinghai Lake[D]. Lanzhou： Gansu Agricultural University， 2013， 61-68.
[3] LI TC. The "starvation effect" phenomena of mineral elements in biology[J]. Qinghai Prataculture， 2015， 24（4）： 2-4.
[4] LI TC. The accumulation differentiation of mineral elements in grassland on the north bank of Qinghai Lake[J]. Qinghai Prataculture， 2015， 24 （3）： 2-6.
[5] MAO DR. Plant nutrition research methods[M]. Beijing： China Agricultural University Press， 1994， 12-21.
[6] XIN YJ. Qinghai grassland resources[M]. Xining： Qinghai People??s Publishing House， 2012， 186.
[7] LIAO H， YAN XL. Advanced plant nutrition[M]. Beijing： Science Press， 2003， 84-241.
[8] SUN X. Plant nutrition and fertilizer[M]. Beijing： China Agriculture Press， 2000， 101-178.
Key words Potted Poa crymophila cv. Qinghai； Mineral element； Accumulation； Liquid mixed fertilizers
Mineral elements are essential nutrients for plant growth and development. Degraded grassland has more accumulation of mineral elements than that of enclosed grassland plants. The Starvation Effect of mineral elements is the endogenetic forces driving the accumulation of grassland mineral elements， and mineral elements are also important responses to grassland succession[1-4]. Is it possible to increase the accumulation of mineral nutrients in alpine grassland plants and soils by spraying liquid mineral mixed fertilizers on a regular basis？ In this paper， potted Poa crymophila cv. Qinghai plants were regularly applied with liquid mineral mixed fertilizers[5]， and samples were collected and analyzed to observe the changes of mineral elements in potted P. crymophila plants and its soil. The aim was to explain the effect of supplementary supply on the nutrient changes of mineral elements in alpine grassland plants. This study was of great significance for the study on the accumulation of mineral elements in degraded grassland plants and its dynamic mechanism， as well as testing the hypothesis of the Starvation Effect of mineral elements. In addition， it could also provide scientific bases and technical support for the restoration and remediation of degraded grassland and the development of ecological livestock husbandry.
Materials and Methods
Test materials
P. crymophila is a species of sparse plexiform grasses with developed roots and stems and strong tillering ability. With upright stems and lush leaves， it can adapt to the complex habitat conditions of the plateau and grow well under different altitude conditions. With the resistances to drought and cold and tolerances to saline??alkaline and barren， it has no strict requirements on the soil. Artificially cultivated P. crymophila grassland can be used as a mowing pasture or served as both mowing and grazing[6]. P. crymophila has soft stems， which taste slightly sweet with good palatability， lush foliage， well??developed vegetative shoots， and a long growing period. After maturity， the branches and leaves remain green and the leaves are not easy to fall off. The crude protein content of leaves in different developmental stages is close to that of alfalfa. Before flowering， the plants are favored by horses， cattle， and sheep. The hay has a soft texture and can be used as a good supplementary feed in winter and spring， and it is also good forage grass for various livestock to gain weights in summers and autumns and to keep the weights in spring and winter[6].
P. crymophila is a kind of grassland plant which is suitable to plant on the Qinghai??Tibet Plateau， and there are commercialized seeds commercially available （the test P. crymophila seeds were provided by De Kejia researcher of the Institute of Grassland of the Academy of Animal Husbandry and Veterinary Science of Qinghai Province）， which are easy to cultivate and have a long growing period. Therefore， P. crymophila is an ideal grassland plant species for grassland plant experiments.
Pot test
The test was conducted in mid??April 2016 in Xining （inside the Northwest Institute of Plateau Biology， Chinese Academy of Sciences）. There were a total of 12 pots of potted P. crymophila （pot diameter of about 30 cm）. The potted soil was grassy black loam （collected from the surrounding pastures around Daban Mountain Haibei Alpine Meadow Localizer Station， mixed with organic fertilizer from cow and sheep manure）， and the seeding rate of P. crymophila was 20 kg/hm2.
Liquid mineral mixed fertilizer： the analytical reagents containing the needed mineral elements were weighed accurately and dissolved in purified water to prepare the liquid mineral mixed fertilizers. The prepared mixed fertilizers contained K of 0.2%， Ca of 0.1%， Mg of 0.04%， Fe of 0.02%， Mn of 0.01%， Zn of 40 mg/L， and Cu of 12 mg/L.
When spraying the liquid mineral fertilizers， the amounts were applied in different gradients， namely， 20 ml/pot， 50 ml/pot and 100 ml/pot， respectively， and the supplement amounts of water were 80 ml/pot， 50 ml/pot and 0 ml/pot， respectively. The control group was irrigated with water of 100 ml/pot. The spraying was done every 10 d from the beginning of May， a total of 10 times. The usual irrigation cycle was 1 time every 1-2 d depending on the weather conditions.
The test lasted until late August， that is， the growth period was 4 months. The above??ground parts of potted P. crymophila were collected according to the test requirements， which were then dried in the shade for later use. Soil samples were prepared by mixing any 3 soils collected randomly from the test pots using a small self??made soil drill， which were also dried in the shade for later use. The collected plant samples were rinsed with deionized water and dried at 60 ??， and the collected soil samples were manually sorted to remove plant roots and other tissues. Then the plant and soil samples were ground and sieved by a 100??mesh sieve. After the pretreatment， the samples were sent to the analysis and testing center of Northwest Institute of Plateau Biology， Chinese Academy of Sciences to perform analysis and testing of various mineral elements.
Analysis test
Analytical instruments： Inductively Coupled Plasma Mass Spectrometer （NexIONTM 350D， PerkinElmer， USA）.
Analysis elements： Ca， Mg， K， Na， P， Cu， Zn， Fe， Mn， and Cr.
Analysis method： standard curve method.
Statistical analysis of data
SPSS20.0 software was used for t test statistical analysis.
Results and Discussion
Mineral elements in potted P. crymophila
The contents of various mineral elements in potted P. crymophila were measured after spraying the self??prepared liquid mineral mixed fertilizers （Table 1 & 2）.
As shown in Table 1 & 2， except Cu， Mn and K， the contents of other mineral elements were reduced to different degrees after spraying the liquid mineral mixed fertilizers. Moreover， the mineral trace elements like Zn， Cr showed significant differences from those of the control （P
In the test， potted P. crymophila plants were more sensitive to the absorption of trace elements than the macroelements， and the trace elements were much easier to accumulate. Compared with the control group， the absorption and accumulation amounts of trace elements in potted P. crymophila were much larger after spraying the liquid mineral mixed fertilizers regularly. For example， the contents of trace elements Cu and Mn in the high fertilization group were increased by 55.2% and 69.2% from the control， respectively， while the contents of the macroelements Ca and P were increased by 4.45% and 5.28%， respectively. According to the hypothesis of the Starvation Effect of mineral elements， the trace elements like Cu， Mn were accumulated in potted P. crymophila because of the insufficient supply during the growth period， and the regular application of liquid mineral mixed fertilizers provided timely supply for most mineral elements in potted P. crymophila， which resulted in no increase of the accumulation. In other words， the accumulation of mineral elements increase due to the lack of timely supply of mineral elements during the growth period. Potted P. crymophila plants were more in need of trace elements during the growing season. In other words， the untimely supply of trace elements in the potted soil environment made the trace elements in potted P. crymophila in the starvation state， in the potting soil environment， and the timely supply of trace elements was more conducive to the growth of potted P. crymophila. There were no P， Cr in the liquid mineral mixed fertilizers. There was no statistically significant difference in the content of P in P. crymophila from the control， while the content of Cr decreased with the increase of spraying amounts， that is， the content of mineral elements in the potted plants of P. crymophila increased due to the application of liquid mineral mixed fertilizers， while there was no significant change in the contents of mineral elements that were not contained in the mixed fertilizers. In other words， the appropriate supply of mineral nutrients could increase the nutrition of the corresponding minerals in the potted plants， which was beneficial to the normal growth of the plants[7-8].
Mineral elements in potted P. crymophila soils
The contents of various mineral elements in potted P. crymophila soils were measured after spraying the self??prepared liquid mineral mixed fertilizers （Table 3 & 4）.
As shown in Table 3 & 4， the contents of mineral elements in the soils were decreased to different degrees after spraying the liquid mineral mixed fertilizers， and showed significant differences from the control （P
On the other hand， the application of liquid mineral mixed fertilizers could also effectively reduce the contents of corresponding mineral elements in potted soils， that is， the application of liquid mineral mixed fertilizers to the potted plants could promote the transportation of more corresponding mineral elements in the soils to the plant tissues （Table 3 & 4）. In other words， compared with the control group， the application of liquid mineral mixed fertilizers could make the potted P. crymophila plants absorb more mineral elements from the soil environment， resulting in the reduction of the corresponding mineral elements in the soils （P
The pot test results showed that the regular supply of liquid mineral elements could reduce the contents of mineral elements in potted plants. According to the Starvation Effect of mineral elements， the regular application （once every 10 d） of liquid mineral mixed fertilizers could make the mineral elements in potted P. crymophila in a state of "non??starvation" due to the timely supply of nutrients， but the potted P. crymophila plants in the control group could not get timely supply， so the mineral elements were accumulated due to the starvation of mineral elements caused by the not timely supply， indicating that the starvation effect was one of the endogenetic forces driving the increase in the accumulation of mineral elements. In the test， the accumulation of mineral elements increased in both the potted plants of P. crymophila and the soils of the control group， while the decrease of mineral elements in both the plants and soils with regular application of the mineral mixed fertilizers （Table 1 & 3， Table 2 & 4）. The supply application of mineral element nutrients could accumulate in the underground root tissues of potted P. crymophila. The results showed that the reduction of mineral elements in potted P. crymophila plants and alpine grassland plants was a timely response and adaptation to the changes in the growth environment， that is， the timely supply of mineral elements could reduce the mineral elements in grassland plants because of the non??starvation state， which was also a phenomenon caused by the Starvation Effect of mineral elements， re??inspecting the hypothesis of the Starvation Effect of mineral elements[1-3].
Conclusion
The regular application of liquid mineral mixed fertilizers can reduce the mineral elements in both the potted P. crymophila plants and soils， suggesting that the supply of mineral elements can effectively increase the applied mineral elements in potted P. crymophila plants， promoting their normal growth. Moreover， it can also promote the transportation of more corresponding mineral elements in the soil to plant issues， thereby reducing the mineral element contents in potted soils.
Compared with the application of liquid mineral mixed fertilizers， the mineral elements are accumulated in the control group since the mineral elements are in a starvation state caused by the untimely supply of mineral elements， indicating that the starvation effect is one of the endogenetic forces driving the increase in the accumulation of mineral elements.References
[1] LI TC. Grassland mineral elements[M]. Beijing： Chemical Industry Press， 2014， 77-83.
[2] LI TC. Study on distribution patterns and accumulation differentiation behavior of mineral elements in grassland on the north bank of Qinghai Lake[D]. Lanzhou： Gansu Agricultural University， 2013， 61-68.
[3] LI TC. The "starvation effect" phenomena of mineral elements in biology[J]. Qinghai Prataculture， 2015， 24（4）： 2-4.
[4] LI TC. The accumulation differentiation of mineral elements in grassland on the north bank of Qinghai Lake[J]. Qinghai Prataculture， 2015， 24 （3）： 2-6.
[5] MAO DR. Plant nutrition research methods[M]. Beijing： China Agricultural University Press， 1994， 12-21.
[6] XIN YJ. Qinghai grassland resources[M]. Xining： Qinghai People??s Publishing House， 2012， 186.
[7] LIAO H， YAN XL. Advanced plant nutrition[M]. Beijing： Science Press， 2003， 84-241.
[8] SUN X. Plant nutrition and fertilizer[M]. Beijing： China Agriculture Press， 2000， 101-178.