Variation in Enzymes Activities of Rhizospheric Substrate and Influencing Factors during Nursing of Watermelon Seedlings

Tingting XUE, Jihua WANG*, Xiaohong SUN

1. College of Life Science and Technology, Harbin Normal University, Harbin 150025, China;

2. Beijing Agricultural Biotechnology Research Center, Beijing 100097, China

Currently, nursery substrate is usually prepared with peat,vermiculite and other materials, of which the costs are relatively high. Even worse, peat is a kind of non-renewable resource, and its mining has been limited. The edible fungus industry has been developed rapidly in China, which has also brought environmental problems. The cultivation waste of edible fungi is usually treated by conventional approaches,such as discarding and burning,resulting in not only waste of resources but also pollution of environment[1-2].A large of experimental studies have shown that mushroom residue, when used as composite nursery substrate,has various advantages. The physical and chemical properties of mushroom residue and its effects on biological indicators(i.e.plant height,stem diameter, shoot dry weight, root dry weight,seedling index) of plants have been studied in previous studies, indicating that mushroom residue is a good potential nursery substrate and it can be used as a replacement for peat to some extent.

Substrate enzyme activity is an important indicator reflecting material and energy metabolism and soil quali-ty, and it has a significant impact on the growth and development of plants and soul fertility. In this study, the composted mushroom residue was mixed with garden soil according to a certain proportion to prepare nursery substrate. The nursery pots were adopted for seedling.The activity variation of urease, alkaline phosphatase,acid phosphatase and neutral phosphatase in the rhizospheric substrates with different formulas was studied,and the correlation between enzyme activity and total nitrogen content,total phosphorus content and organic matter content in certain substrate was investigated so as to explore nutrients composition and enzymes activities in nursery substrate and the mutual influences and restrictions between substrate enzyme activity and nursery.

Table 1 Formula of mushroom residue composite substrate(volume ratio)

Table 2 Correlations between pH value and fertility factors of substrate and urease and phosphatases activities in rhizospheric substrate

Table 3 Correlations between shoot and root dry weights and enzymes activities at different growth stages

Materials and Methods

Materials

The mushroom residue used in this study included composted shiitake mushroom residue and oyster mushroom residue.The garden soil was collected from the test field of Vegetable Technical Service Center of Shunyi District, Beijing City, and the pig manure was collected from a farmer’s piggery in Beijing. The watermelon cultivar was Xiaohongshuai.

Test design

The watermelon seedlings were all nursed in nursery pots. A total of 4 treatments were designed (Table 1), and there were 5 replicates for each treatment. The organic fertilizer(soil ∶pig manure=3 ∶1) was used as control.

Indicator measurement

The physical and chemical properties of the substrate were studied 10, 20 and 30 d respectively after the emergence of watermelon seedlings.The measured indicators included pH value, total nitrogen content, total phosphorus content, shoot dry weight,root dry weight and activities of enzymes (urease, neutral phosphatase,acid phosphatase, alkaline phosphatase)in rhizospheric substrate.

Methods

Biological indicators The measured biological indicators included shoot dry weight and root dry weight(electronic balance).

pH For pH measurement, a certain amount of substrate was dissolved in a certain volume of water(m∶v, 1∶5) and then mixed by shaking for 1.5 h. Subsequently,the mixture was centrifuged at 10 000 r/min for 10 min. Then the pH value of the supernatant was measured by pH meter[3].

Enzymes activities The rhizospheric soil of the watermelon seedlings was sampled on day 10, 20 and 30 since the emergence of watermelon seedlings. A total of 5 watermelon seedlings were selected for each replicate of each treatment. The seedling roots were dug out carefully,and the soil on the roots surface was shaken off gently. The soil collected from each replicate was mixed together and placed in a soil collection bag.The soil was passed through a 20-mesh sieve and preserved at 4 ℃for enzyme activity measurement. The activities of 4 kinds of enzymes, including urease, neutral phosphatase,acid phosphatase and alkaline phosphatase were measured.The activities of the phosphatases were measured by Hoffinan method[4], and the activity of urease was measured by urea colorimetric assay[5].

Total nitrogen content and total phosphorus content The total nitrogen content in the mushroom residue composite substrate was determined using international standard method[6]. The soil was first dissolved in certain solvent, and then the total nitrogen content was determined. The total phosphorus content was also determined using international standard method[7].

Data analysis

The obtained data was processed using Microsoft Excel (Office 2007)and analyzed using SPSS(IBM SPSS Statistics 19.0).The drawing was performed using OriginPro8.5.

Results and Analysis

Variation in pH value of rhizospheric substrate

As shown in Fig.1, with the proceeding of the test, the pH values in the substrates were overall trended to be decreased.The pH value in the M3 substrate was highest, followed by those in the M2 and M1.The substrate in the control group was weakly acidic.The variation of pH value in the rhizo-spheric substrate, with the growth of watermelon seedlings, was also studied. The results showed that with the growth of seedlings, the pH value in the rhizospheric substrate was reduced gradually. With the growth of seedlings,the root scale was enlarged gradually. Thus the secreted organic acids by roots and produced carbonic acid,formed by CO2that was secreted by respiration were all enhanced. This explained why the pH value in rhizospheric substrate declined gradually with the growth of watermelon seedlings.

Variation of total nitrogen content in rhizospheric substrate

The nursery substrate provides nutrients for watermelon seedlings.So the nutrients contents in substrate will directly determine the normal growth of seedlings. Soil total nitrogen includes all forms of soil organic and inorganic nitrogen, and it reflects the nitrogen status of the soil[8]. The rhizospheric substrate was sampled on day 10, 20 and 30 since the emergence of watermelon seedlings. The total nitrogen contents in the substrate samples were determined.As shown in Fig.2,in the first 10 days,the total nitrogen contents in the rhizospheric substrates were decreased gradually; there were no obvious changes in total nitrogen contents from day 10 to day 30.It indicates that the nitrogen in substrate has basically been exhausted in the early growth period of seedlings,and a right amount of exogenous nitrogen should be supplemented for maintaining the normal growth of seedlings.

Variation of total phosphorus content in rhizospheric substrate

As shown in Fig.3,the total phosphorus contents were reduced rapidly from 0 to day 10. Similarly, in the later 20 days,the total phosphorus contents showed no significant changes. It indicates that the phosphorus in substrate has basically been exhausted in the early growth stage of seedlings, and a right amount of exogenous phosphorus should be supplemented timely for maintaining the normal growth of seedlings.

Variation in enzymes activities of rhizospheric substrate

Urease Fig.4 showed that the urease activity in the rhizospheric substrate was determined by total nitrogen content and number of microorganisms in the substrate. The total nitrogen content in the substrate was reduced gradually with the growth of the seedlings, but the number of rhizospheric microorganisms and root dry weight was increased greatly.With the growth of the watermelon seedlings,the urease activity in the rhizospheric substrate was trended to be decreased, indicating that the total nitrogen content in the substrate played a key role in determining the urease activity.

Alkaline phosphatase As shown in Fig.5, in the whole nursery stage of watermelon seedlings, the alkaline phosphatase activity was first increased and then decreased in the substrates CK and M1, but it was first decreased and then increased in the substrates M2 and M3. Among the 4 kinds of substrates, the alkaline phosphatase activity was highest in M3,followed by those in M2 and CK, and the alkaline phosphatase activity was lowest in M1. On day 20, the alkaline phosphatase activity in the composite substrate was increased naturally,which first occurred in the substrates M1 and CK. The M1 substrate was composed of oyster mushroom residue, shiitake mushroom residue and garden soil (v∶v∶v,1∶1∶2),and the CK substrate was composed of garden soil and pig manure(v∶v,3∶1).The mushroom residue and organic manure could improve the alkaline phosphatase activity, but with the decomposition of composite phosphorus and organic matters in the mushroom residue and manure, the alkaline phosphatase activity was reduced naturally. The M2 and M3 substrates were all weakly alkaline, so their alkaline phosphatase activities were relatively high. The activity of secreted phosphatase in the composite substrate M3 was relatively high, so the alkaline phosphatase activity in the rhizospheric substrate of M3 was increased correspondingly.

Neutral phosphatase Fig.6 showed that in the early growth stage of watermelon seedlings, none obvious regularity was found in variation of neutral phosphatase activity in all the 4 kinds of substrates. However, in the late growth stage,the neutral phosphataseactivity was trended to be decreased,indicating that the reduced total phosphorus content in substrate played the dominant negative effect on neutral alkaline phosphatase activity. However,during the nursing of muskmelon seedlings,the neutral phosphatase activity in rhizospheric substrate was increased gradually with the growth of seedlings,indicating that the increased number of microorganisms was the main factor for increased neutral phosphatase activity.

Acid phosphatase Fig.7 showed the effect of different composite substrate on variation of acid phosphatase activity in rhizospheric substrate with the growth of watermelon seedlings.In the whole nursery stage, the acid phosphatase activities in different substrates were all increased first and then decreased. On day 10, the acid phosphatase activity in the composite substrate M1 was highest, followed by those in M2 and M3, and the acid phosphatase activity in the substrate CK was lowest. On day 20, the acid phosphates activity was highest in M3,the enzyme activity in M1 was a little higher than that in M2, and the enzyme activity in CK was lowest. On day 30, the acid phosphatase activity was highest in M1, followed by those in M3 and M2, and the acid phosphatase activity was lowest in CK.The substrate in the CK treatment group was weakly alkaline,so the acid phosphatase activity in the rhizospheric substrate was relatively low. The acid phosphatase activity was positively related to total nitrogen content in substrate, so the variations of total nitrogen content and acid phosphatase activity in the substrates were consistent with the growth of watermelon seedlings.

Correlation analysis between physical and chemical properties of rhizospheric substrate and enzymes activities

Correlation between pH value of substrate and enzymes activities As shown in Table 2, the 4 kinds of substrates were all weakly alkaline.The correlation analysis between pH value and phosphatases activities showed that there was no significant correlation between pH value and urease activity (r=0.566); the pH value was significantly correlated to alkaline phosphatase activity (r =0.875); the correlation coefficients between pH value and neutral and acid phosphatase activities were 0.667 and 0.486, respectively. The effects of pH on soil enzymes activities are mainly realized though affecting microbial activity in soil. This study found that the pH value of alkaline substrate was positively correlated to alkaline phosphatase activity, which was consistent with previous studies[8].

Correlation between fertility factors and enzymes activities The correlations between substrate nutrients and urease and phosphatases activities were analyzed (Table 2). The neutral phosphatase activity was significantly correlated to total phosphorus content(r=0.923,P＜0.01)and organic matter content (r=0.317, P ＜0.05). In the mushroom residue composite substrates, the urease activity was significantly correlated to organic matter content (r=0.823,P＜0.01)and total nitrogen content (r=0.798, P ＜0.01); the urease was closely related to nitrogen transformation in substrate,and the urease activity in substrate could reflect the nitrogen supplying level and capacity of substrate[9].

Correlation between root dry weight and activities of urease and phosphatases The correlations between shoot and root dry weights and enzymes activities were studied (Table 3). The results showed that the phosphatases activities were positively related to root dry weight (P＜0.01),and the correlation coefficients between phosphatases activities and root dry weight at the three growth stages were 0.724*, 0.536*and 0.774**, respectively. In different nursery substrates,the higher the root dry weight is, the higher the phosphatases activities are.There were no significant correlations between root dry weight and urease activity with correlation coefficients of 0.391,0.322 and 0.670 respectively at the three growth stages. However, the urease activity was positively related to shoot dry weight(P＜0.01).The correlation coefficients between urease activity and shoot dry weight at the three growths stages were 0.724*,0.698*and 0.997**, respectively. In different nursery substrates,the higher the shoot dry weight is, the higher the urease activity is. There were no significant correlations between phosphatases activities and shoot dry weight[10]. The correlation coefficients between shoot dry weight and phosphatases activities at the three growth stages were 0.515, 0.363 and 0.448,respectively.

Conclusions

With the growth of watermelon seedlings,the pH value in rhizospheric substrate was reduced gradually, and the urease activity in rhizospheric substrate was trended to be de-creased. Among the 4 kinds of substrates, the alkaline phosphatase activity was highest in the substrate M3.

During the nursing of watermelon seedlings, the urease activity in rhizospheric substrate was positively related to shoot dry weight and total nitrogen content; the neutral phosphatase activity was positively related to root dry weight and total phosphorus content; the alkaline phosphatase activity was positively related to pH value in rhizospheric substrate.

In the first 10 d since the emergence of watermelon seedlings, the total nitrogen content and total phosphorus content were reduced rapidly;the total nitrogen content and total phosphorus content showed no significant changes from day 10 to day 30.It suggested that the nitrogen and phosphorus in the substrate had basically been exhausted in the first 10 d since the emergence of watermelon seedlings, and right amounts of exogenous nitrogen and phosphorus should be supplemented timely to maintain the normal growth of seedlings.

[1]LIU WW (刘雯雯),YAO T (姚拓),SUN LN (孙丽娜). The feasibility of spent mushroom substrate as a kind of microbial fertilizer carrier (菌糠作为微生物肥料载体的研究)[J]. Journal of Agro-Environment Science(农业环境科学学报),2008,27(2):787-790.

[2]XIE JL(谢嘉霖),LIU RH(刘荣华),YE QF(叶启芳), et al. Researches into media EC and determination conditions (无土栽培基质电导率和pH 值测定条件的研究)[J].Journal of Anhui Agricultural Sciences (安徽农业科学), 2006, 34(3):415-416.

[3]ZHAO LP(赵兰坡),JIANG Y(姜岩).Discussion on methods for determination of soil phosphatase activity(土壤磷酸酶活性测定方法的探讨)[J].Chinese Journal of Soil Science(土壤通报),1986,17(3):138-141.

[4]LI FD(李阜棣).Soil Microbiology(土壤微生物学)[M]. Beijing: China Agriculture Press(北京: 中国农业出版社),1996.

[5]National Bureau of Standards(国家标准局). GB 7173-1987 Method for the determination of soil total nitrogen (semimicro Kjeldahl method)(土壤全氮测定法(半微量开氏法)).Beijing:Beijing Agricultural University(北京: 北京农业大学),1987.

[6]Ministry of Agriculture of the People's Republic of China(中华人民共和国农业部).GB 9837-88 Method for determination of soil total phosphorus(土壤全磷测定法(碱熔-钼锑抗分光光度法)).Beijing: Chinese Academy of Agricultural Sciences(北京: 中国农业科学院),1988.

[7]STAMPS RH, MR EVANS. Growth of Dieffenbachia maculate Camille. in growing media containing sphagnum peat or coconut coir dust[J].Horticulture Science,1997,32(5):844-847.

[8]JONER EJ, JAKOBSEN I. Growth and extracellular phosphatase activity of arbuscular mycorrhizal hyphae as influenced bysoil organic matter [J].Soil Biology and Biochemistry, 1995, 27(6):1153-1159.

[9]ZHU LIXIA, ZHANG JIA EN. Review of studies on interactions between root exudates and rhizospheric microorganisms[J].Ecol Environ,2003,12(1):102-105(in Chinese).

[10]JAW M, BENDING GD, WHITE PJ.Biological costs and benefits to plantmicrobe interactions in the rhizosphere[J]. Journal of Experimental Botany,2005,56:417-418.