Chong YUE, Baoguo FAN

Colleage of Life Science, Shanxi Normal University, Linfen 041000, China

Allelopathy effect of plants refers to the phenomenon that a living plant through releasing allelochemicals produced itself into environment influences the growth and development of surrounding plants,including promotion and inhibition effects[1]. In some researches, it was found that garlic could be a kind of good preceding crop for a lot of vegetable crops[2],the allelochemicals produced by wild mustard could improve the yield of cauliflower[2], while the allelopathy effects of most plants exhib ited as a harmful inhibition effect,which not only existed between plants,such as Rehmannia glutinosa[3],Artemisia annua Linn.[4], Eucalyptus robusta Smith[5], but also caused in agricultural production serious continuous cropping obstacle on some crops, for example, the continuous cropping of strawberry[6], cucumber[7],soybean[8], and pea[9]caused yield reduction and heavier disease.

Adzuki bean (Phaseolus angularis) is a kind of coarse cereals with quite high nutritional value,whose culture area increases year by year due to market demand, but owing to the constraint of crop rotation in culture,the yield and quality of adzuki bean has been influenced greatly. Due to continuous cropping obstacle of adzuki bean, wheat is chosen to be the rotation crop for adzuki bean in some areas, though the reason has not been reported.It was found that in the some areas of the Loess Plateau,due to the restriction by climatic conditions, the field experienced the plantation of adzuki bean is often left uncultivated,resulting in serious waste in land resource. Radish and cabbage are im-portant vegetables in Cruciferae, having high nutritional value and medicinal value, and therefore, with a continuously increased demand, it is very important to realize high-quality production and balanced supply of radish and cabbage.

In this study, adzuki bean root aqueous extract was used as the study object,from an allelopathy effect point of view,the reason of continuous cropping obstacle of adzuki bean and the effects on different receptor crops were investigated by indoor petri dish bioassay, which was of great importance in selection of rotational crop,rational utilization of land resources,protection of production region,etc.

Materials and Methods

Materials

The test was carried out during November 6 -28 in 2013 at Shanxi normal university. The test adzuki bean variety was ‘Bao 8824-17’.The root aqueous extract was prepared by cleaning adzuki bean root,grinding after air drying, weighing 100 g into a beaker (1 l)and adding distilled water as extraction regent. The extraction was performed at room temperature for 72 h, followed by filtration and centrifugation (2 500 r/min, 4 ℃) for 10 min. The obtained supernatant was added with distilled water to 1 l to get a mother solution with a concentration of 100 g/L. The mother solution was diluted to three concentrations of 20, 10 and 5 g/L, and the dilutions was bottled for later use. The receptor crops were ‘adzuki bean Bao 8824-17’,‘Jinmai 1718’, ‘radish Teda 791’and‘Yihe Qiubaicai’.

Test design

The root aqueous extract of ‘Bao 8824-17’ was provided with four levels, 20, 10, 5 and 0 g/L (CK). plump seeds of radish, cabbage, wheat and adzuki bean with substantially the same size were selected for the test.For each treatment, 100 seeds were sown into petri dishes laid with two layers of filter paper.The root aqueous extracts with concentrations of 20, 10,and 0 g/L were added, respectively,while distilled water was added into the control. Culture was performed in an artificial climate incubator. In the germination stage, 5 ml of adzuki bean aqueous extracts were added in the morning daily while 5 ml of distilled water were added in the afternoon to maintain the dishes moist. After germination, 1/2 Hoagland nutritive medium was added to maintain normal growth of plants. Each treatment was repeated for three times. The number of germinated seeds in each dish was recorded, and the morphology and physiological indexes of seedlings was determined after 15 days.

Index determination and methods

Germination index: The number of seeds germinated on the right day was recorded at 10 a.m. daily, considering the seeds with radicle lengths not shorter than the lengths of seed diameters as germinated seeds. The germination was completed when the number of germinated seeds did not increase in 2 days continuously. Germination rate (G),germination index and inhibition ratio(IR) were calculated.

The inhibition ratio(IR)was calculated according to Lin et al.[11],in which an inhibition effect existed when IR≥0, while a promotion effect existed when IR＜0, and the absolute value of IR represented the strength of the effect.

Gt was the number of germinated seeds at tth day; Dt was the germination days(until the day with the largest number of germinated seeds).

Seedling growth indexes: plant height, radicle length, spire height, lateral root number, single-plant fresh mass.

Physiological index determination: SOD activity was determined according to nitroblue tetrazolium (NBT)method, POD activity was determined according to guaiacol method,and MDA content was determined according to thiobarbituric acid (TBA)method[12].

Statistic analysis of data

Experimental data were processed with Excel 2007, and variance analysis and Duncan multiple comparison were performed with DPS.

Results and Analysis

Influences of adzuki bean root aqueous extract on germination of 4 kinds of receptor crop seeds

Influence on germination of adzuki bean seeds From Table 1, in comparison with the control, the seeds of adzuki bean ‘Bao 8824-17’ had an obviously decreased germination rate after being treated with the adzuki bean root aqueous extracts, and the difference reached significant level.The germination index and germination rate were both on the decrease with the increasing aqueous extract concentration. When the aqueous extract had a concentration of 5 g/L, the inhibition ratio of the germination index and the inhibition ratio of germination were of 59.85%and 54.55%,respectively;when the aqueous extract had a concentration of 10 g/L, the inhibition ratio of the germination index and the inhibition ratio of germination were of 66.43%and 60.61%,respectively;and when the aqueous extract had a concentration of 20 g/L, the inhibition ratio of the germination index and the inhibition ratio of germination were of 90.02% and 81.82%, respectively. It was thus clear that the root aqueous extract of adzuki bean had an inhibition effect on the germination of its seeds, and furthermore, with the increase in the concentration of the aqueous extract, the inhibition effect was strengthened, indicating that adzuki bean had a very strong autotoxicity. The autotoxicity might be the main obstacle for continuous cropping of adzuki bean.

Influences on germination of the seeds of other 3 kinds of receptor crops From Table 1 also, the seeds of wheat had decreased germination rates after being treated with the adzuki bean root aqueous extracts with different concentrations, though the difference with the control was not significant under low concentrations,which was then reached significant level at the concentration was of 20 g/L;the germination rate of radish also decreased, but was not significantly different with the control,and especially, the inhibition ratio was only of 6.56% at the concentration of 20 g/L,still with a non-significant difference;and a decrease also happened on the germination of cabbage seeds, while the difference with the control was significant,with a highest inhibition ratio of 19.51%.

The influences of adzuki bean root aqueous extract on the germination indexes of tested seeds were more obvious those on germination rates, and it thus could be seen that the aqueous extract prolonged the germination time of seeds, influenced the germination process of seeds and obviously decreased the germination index. Among the three crops, cabbage suffered most obvious inhibition effect with an inhibition ratio up to 66.40%,and the germination index of wheat had an inhibiting extent less than it,with an inhibition ratio up to 56.31%.However, the inhibition suffered by them showed different laws,the former one suffered a continuously aggravated inhibition effect, while the inhibition extent of the later one was light at first and serious then. On the contrary, the treatment with low concentration aqueous extract (5 g/L) improved the germination index of wheat to different extents, but such positive allelopathy effect gradually decreased with increasing concentration of the treatment solution. In practical production,wheat serves as the crop cultivated after the harvest of adzuki bean. It could be seen from the germination rate and germination index that radish and cabbage also could be cultivated after adzuki bean.

Table 1 Influences of adzuki bean root aqueous extract on germination of 4 kinds of receptor crop seeds

Table 2 Influences of adzuki bean root aqueous extract on germination of 3 kinds of receptor crop seedlings

Influences of adzuki bean root aqueous extract on seedlings of the 3 kinds of receptor crops

Influences on growth of overground parts of seeldings of the 3 kinds of receptor crops With seedling growth time, the seedlings of adzuki bean could not grow normally under the allelopathy effect of adzuki bean aqueous extract. In Table 2, the plant heights and spire lengths of radish and cabbage both were inhibited at the low concentration, but were not significantly different with those of the controls; and the plant height of wheat was inhibited at 5 g/L, but was not significantly different with that of the control. When the concentration was of 10 g/L, the plant height of wheat was promoted.The spire length of wheat was in positive correlation with the concentration of the aqueous extract, indicating that the root aqueous extract of adzuki bean helped the growth of the overground part of wheat.

Influences on growth of underground parts of seeldings of the 3 kinds of receptor crops The laws of the allelopathy effects of adzuki bean root aqueous extract on radicle lengths and lateral root number of seedlings were different. From Table 2, when the concentrations were of 5 and 10 g/L,the inhibition rates of lateral roots of radish were of 3.28% and 27.87%, respectively, and the inhibition rates of lateral roots of cabbage were of 9.46% and 35.14%, respec-tively. It was thus indicated that with increasing aqueous extract concentration, the elongation of radicles of radish and cabbage was significantly inhibited, and the number of lateral roots was reduced. However, the differences between the radicle lengths and lateral root numbers of seedlings of radish and cabbage were not significant.On the contrary,wheat exhibited a positive allelopathy effect which was strengthened with the increasing concentration.

Influences on fresh masses of the 3 kinds of receptor crops It also could be known from Table 2,the fresh masses of radish and cabbage both exhibited a low-concentration promoting and high-concentration inhibiting trend with increasing concentration of adzuki bean root aqueous extract. Under the treatment by extracts with different concentrations,the fresh masses of radish seedlings were significantly different; and the fresh mass of cabbage seedling presented a minimum at 10 g/L, which was decreased by 19.23%and 22.22%respectively in comparison with those at 0 and 5 g/L,but such changes were not significant.Adzuki bean root aqueous extract had a positive allelopathy effect on the fresh mass of wheat seedlings. At 10 g/L, the inhibition rate was the lowest with an IR index of -44.00%, and a significant difference existed in comparison with the control.

Influences on physiological indexes of the 3 kinds of receptor crops

As shown in Fig.1,the SOD activity of both radish and wheat exhibited a trend of increasing at first and decreasing then, they reached their greatest SOD activity at 5 g/L, which was higher than the controls by 8.10%and 9.37% , and at the concentration of 10 g/L, their SOD activity began to decrease, but was not significantly different with that of the controls;while for cabbage,the SOD activity was significantly decreased with increasing allelopathic substances,indicating that the amount of existing allelochemicals has already exceeded the tolerance of cabbage seedlings, and cabbage was thus inhibited.

It was shown in Fig.2 that at the low concentration(5 g/L),the POD activity of seedlings of radish and wheat increased slowly with the increase of the concentration of adzuki bean root aqueous extract, indicating that 5 g/L adzuki bean root aqueous extract could start POD protection effect; and as to cabbage, the POD activity was on the decrease, with a greatest decrease amplitude of 57.66% from the control to the low concentration(5 g/L),which was significant.The decrease in POD activity of cabbage seedlings indicated that the physiological metabolism had been significantly influenced.

Fig.3 showed that the MDA contents of the 3 kinds of seedlings had different changes with increasing aqueous extract concentration. The MDA contents of cabbage at 5 and 10 g/L were obviously increased in comparison with the control, which were 1.50 and 7.66 times of that of the control. The MDA contents of radish and wheat both decreased at first and then increased,which might due to that low concentration root aqueous extract of adzuki bean activated the protection enzymes in radish and wheat, inhibiting peroxidation of membrane lipid resulting in low MDA yield,but with growing inhibition effect, overall protection ability of the protection enzyme system decreased, and subsequently, MDA content increased under high concentration,which was not significantly different with that of the control. It was thus indicated that radish and wheat had good adaptability capable of resisting allelopathic inhibition effect by adzuki bean, and could serve as the crop cultivated after adzuki bean.

Conclusions and Discussion

Autotoxicity and allelopathy effect of plants are universal[13-16]. Serious obstacle has found in continuous cropping of adzuki bean.The results of this paper showed that: increasing concentration of adzuki bean root aqueous extract strongly inhibited its own germination rate and germination index, i.e., it was of concentration effect type. This research result was substantially similar to the experimental results from other plants[18-19], and the autotoxicity of adzuki bean was one of the main reasons for the continuous cropping obstacle.

Allelochemicals first influence germination of seeds, determine seeds to germinate or not, or determine seedlings to be healthy or not,and thereby influence the growth of receptor plants. Therefore, germination rate, germination index and seedling growth are often considered as the indicators for evaluating the sensitivity of plants to allelopathic effect.

The allelpathic inhibition effect of adzuki bean root aqueous extract to germination of radish and cabbageseeds had an overall law that the allelopathic inhibition effect was gradually enhanced with increasing aqueous concentration. The germination rate and germination index of seeds of each kind of plant were inhibited to different extents, but inhibition extents of the 3 kinds of receptors were not significant in comparison with the control;and the allelopathic effect of adzuki bean to cabbage seeds was not mainly in inhibiting germination rate thereof but in decreasing seed activity, mainly resulting in delayed germination,which was the same as the result obtained by Geng et al.[20]. The influence of allelopathic effect on seed germination could only be reflected accurately and comprehensively by comprehensively using germination rate and germination index[21].

The determination results of the 3 kinds of receptor crop seedlings showed that the growth states of receptor plants were influenced by the adzuki bean root aqueous extract to different extent compared with the controls. Radish and cabbage had their various morphological characteristics subjected to weaker inhibition effect. Wheat showed a positive allelopathy effect: the number of lateral roots and plant fresh mass were significantly different with those of the control, indicating that with the increase of aqueous extract concentration, wheat increased root absorption area by increasing lateral root number,to increase plant absorption amount objectively,exhibiting in significant difference between fresh masses of treated plants and the control.

There were researches suggesting that destroying membrane structure and function was one of the mechanisms of allelopathy effect by allelochemicals,and might be the start of various allelopathy effects[22]. With increasing concentration of adzuki bean root aqueous extract, the SOD and POD activities of radish and wheat seedlings increased, leading to decrease in MDA contents of wheat and radish seedlings; while the SOD and POD activities of cabbage decreased significantly,accompanied by increase of MDA content of cabbage by a large amplitude, indicating that cytomembranes of cabbage seedlings were seriously damaged and normal intracellular enzyme protection system was destroyed. Han et al.[23]found similar result from the research on allelopathy effect of ginger to soybean. It was shown that radish also could serve as the rotation crop suitable for adzuki bean besides wheat.

Allelochemicals also influenced crop growth indirectly by influencing soil microorganism group, soil chemical property and nutrient cycling except acting on adjacent crops directly[24-25]. Aiming at how to select the succession crop for adzuki bean,effort still should be made to deeply analyze the cultivation mode of adzuki bean through further soil pot culture test and field test.

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