Allelopathic interference of six alfalfa varieties at different harvesting stages on the germination, seedling and root growth of green foxtail and barnyardgrass

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Allelopathic interference of six alfalfa varieties at different harvesting stages on the germination, seedling and root growth of green foxtail and barnyardgrass

https://doi.org/10.21203/rs.3.rs-4300128/v1

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In order to evaluate the allelopathic interference of different varieties of alfalfa on notorious weeds, the sensitivity of green foxtail and barnyardgrass to the extracts from the first, second, and three stubbles of six varieties were investigated through plate culture. The germination rate, seedling length, seedling fresh weight, root length, and root fresh weight of weeds were measured in this study to comprehensively elucidate allelopathic effects. The results suggested that: (1) The allelopathy of six alfalfa varieties tested showed obvious interspecific differences, the inhibition of Zhongmu No.3 on weeds was weaker than other cultivars. (2) The inhibitory effect of alfalfa extracts on green foxtail was enhanced with the increase of stubble times, while the differences between three stubble times on barnyardgrass were not clear, especially between the first and second stubbles. (3) Compared with barnyardgrass, green foxtail was generally more susceptible to the extracts. (4) The inhibitory effect of alfalfa extract on root was stronger than seedlings in the same weeds. The study may help to comprehensively reveal the allelopathic effect of different alfalfa varieties in the first three stubbles on green foxtail and barnyardgrass, providing scientific evidence for weed control based on natural plant extracts in the future.

Biological sciences/Plant sciences/Plant symbiosis

Biological sciences/Plant sciences

alfalfa

allelopathy

barnyardgrass

green foxtail

weed control

Allelopathy refers to an interference mechanism in which the plant release bioactive metabolites that have direct or indirect beneficial or harmful effects on surrounding plants¹. Most allelopathic effects are suppressed on surrounding plants by releasing phytotoxic substances². Many studies have shown that alfalfa (Medicago sativa) has the certain allelopathic potential to control weeds^3–7. A total of 15 cases of herbicide-resistant weeds have been reported in alfalfa fields globally since 1984, involving 10 types of weeds⁸, all of which pose greater challenges to the chemical control of weeds in alfalfa fields. However, allelopathy of plants might provide an alternative mechanism for weed control that reduces dependence on herbicides, especially for herbicide-resistant weeds.

In fact, there have been many reports on the allelopathic effects of the alfalfa on other weeds. For example, the alfalfa extracts significantly affected seedling growth and root morphology of barnyardgrass (Echinochloa crus-galli)³. Alfalfa with different genotypes displayed a range of allelopathic interference in annual ryegrass (Lolium rigidum) seedlings, reducing the length of roots from 5–65%⁴. The germination and seedling growth of various weed species were inhibited by aqueous extracts from dried alfalfa, including lambsquarters (Chenopodium album), giant foxtail (Setaria faberii), velvetleaf (Abutilon theophrasti), pigweed (Amaranthus retroflexus), crabgrass (Digitaria sanguinalis), cheatgrass (Bromus secalinus) and mugwort (Artemisia vulgaris)^5–6. In addition, the alfalfa leaf extracts inhibited the growth of the calluses of crabgrass, lambsquarters, purple amaranth (Amaranthus lividus), purslane (Portulaca oleracea), and asiatic dayflower (Commelina communis)⁷. However, little was known about the allelopathic interactions between other weeds and different alfalfa varieties with different stubble times.

In this study, the allelopathic potential of six excellent alfalfa varieties suitable for planting in northern China to malignant weeds green foxtail and barnyardgrass was evaluated by measuring the 1) germination, 2) seedling length, 3) seedling fresh weight, 4) root length, and 5) root fresh weight of weeds under the treatment of the first three stubbles extracts. Based on the above five indicators, synthetical allelopathic effect (SE) of methanol extracts from the first three stubbles of six alfalfa cultivars on two types of weeds was also calculated.

Allelopathic effect of alfalfa extracts on the germination of green foxtail and barnyardgrass. There was obvious difference in the allelopathy to the germination of green foxtail between different varieties and different stubble times of alfalfa extracts (Fig. 1-A, B, C). It was clear that the germination rate of green foxtail decreased with the increase of the concentration of the extracts. For the extracts of the first stubble of WL319HQ, the germination rate of 0.05 g mL^− 1 and 0.07 g mL^− 1 group were 46.67% and 22.00%, respectively, significantly lower than 74.00% of the control (Fig. 1-A). Besides, the concentrations of extracts from different alfalfa varieties that significantly inhibited the germination were also unequal. For the first stubble of alfalfa, when the concentration of extracts from Baoding varieties was 0.01 g mL^− 1, the germination of green foxtail was significantly inhibited, while the extracts from Zhongmu No.3 did not inhibit them until 0.07 g mL^− 1 (Fig. 1-A).

Similarly, the allelopathic effects of methanol extracts from different varieties of alfalfa on the germination of barnyardgrass were shown in Fig. 1 (D, E, F). Compared with the control group, the low concentration of alfalfa extracts had almost no effect on the germination of barnyardgrass, while only the high concentration inhibited them. Except for Zhongmu No.3, the other five varieties significantly inhibited the germination of barnyardgrass at 0.07 g mL^− 1 in the matter of the first stubble of alfalfa extracts, while WL525HQ and Medoc Max exhibited significant suppression when treated with the lower concentration (0.03 g mL^− 1) (Fig. 1-D). In addition, only the extracts from the first and third stubble of WL319HQ alfalfa significantly inhibited the germination (Fig. 1-D, F), while the second stubble did not (Fig. 1-E).

Allelopathic effect of alfalfa extracts on the seedling of green foxtail and barnyardgrass. The length and fresh weight of seedlings were measured to evaluate the allelopathy of alfalfa extracts on the overground part of weeds (Fig. 2-A, B, C; Fig. 3-A, B, C). The inhibitory effects increased as the concentration of the alfalfa extracts increased. It was worth noting that the third stubble extracts of all varieties significantly promoted the growth of seedling fresh weight at the lowest concentration (0.01 g mL^− 1) (Fig. 3-C). However, high concentration of extracts could inhibit the fresh weight or the length of green foxtail. For example, the fresh weight inhibition rate caused by extracts from the third stubble of WL343HQ at the concentration of 0.03 g mL^− 1 was 83.75% (Fig. 3-C). Except WL319HQ, the third stubble extracts of the other five varieties completely inhibited the growth of seedlings at the concentration of 0.05 g mL^− 1 (Fig. 2-C, Fig. 3-C), and the second stubble extracts required 0.07 g mL^− 1 (Fig. 2-B, Fig. 3-B). Therefore, it was clearly concluded that the inhibitory effect of the third stubble of alfalfa extract was strongest in three stubbles.

The allelopathy of alfalfa extracts on barnyardgrass seedlings was also determined (Fig. 2- D, E, F; Fig. 3- D, E, F). Compared with green foxtail, the promotion of low-concentration extracts on the growth of barnyardgrass was not uniform. Only the extracts from the first stubbles of WL525HQ and Medoc Max could promote the fresh weight at the lowest concentration (Fig. 3-D). Seedling growth of barnyardgrass was less inhibited by alfalfa extracts than that of green foxtail. The concentration of extracts to inhibit the fresh weight of barnyardgrass was 0.5 or 0.7 g mL^− 1, which was generally higher than that required by green foxtail (Fig. 3). Moreover, the extracts of the second stubbles exhibited the weakest inhibitory effect on barnyardgrass, only WL319HQ obviously inhibited the fresh weight at the highest concentration, and the other five varieties did not (Fig. 3-E).

Allelopathic effect of alfalfa extracts on the root of green foxtail and barnyardgrass. Accordingly, the length and fresh weight of root were used to evaluate the allelopathy of alfalfa extract on the underground part of green foxtail and barnyardgrass (Fig. 4–5). The root of green foxtail and barnyardgrass were more susceptible to the alfalfa extracts than the germination or the growth of seedling. Most of extracts significantly inhibited the length and fresh weight of root of green foxtail at the lowest concentration (Fig. 4-A, B, C; Fig. 5-A, B, C). The concentration of few extracts that significantly inhibited root growth increased to 0.03 g mL^− 1, such as the second stubble of WL343HQ (Fig. 4-B).

The allelopathic effect of alfalfa extract on the roots of barnyardgrass also was accurately measured (Fig. 4-D, E, F; Fig. 5-D, E, F). In general, the allelopathic effects of Zhongmu No.3 and Baoding on the growth of roots were relatively weaker than the other four varieties. As regard to the extract of the first stubble of Zhongmu No.3, the fresh weight of barnyardgrass was not significantly inhibited until the highest concentration (Fig. 5-D). The concentration of the extracts from the second and third stubbles of Zhongmu No.3 that began to inhibite the fresh weight of barnyardgrass was 0.05 g mL^− 1 or 0.03 g mL^− 1, respectively, which illustrates once more the allelopathy enhanced with the frequency of harvests (Fig. 5-E, F).

The SE of the alfalfa extracts on green foxtail and barnyardgrass. The values of SE were acquired by all indicators of green foxtail or barnyardgrass treated with extracts from each alfalfa variety (Table 2–3). In general, the absolute values of SE from Medoc Max on green foxtail were higher than those of Zhongmu No.3 and WL319HQ, indicating that various inhibitory effects existed in different alfalfa varieties (Table 2). As the number of harvests increases, the SE absolute values to green foxtail of alfalfa extracts from each variety increased. To be specific, the absolute values of SE in the third stubble of alfalfa extract for each variety were generally higher than that in the first and second stubbles (Table 2).

Table 2

Synthetical allelopathic effect (SE) of methanol extracts from the first three stubbles of six alfalfa cultivars on green foxtail.
Variety	SE
Variety	First stubble	Second stubble	Third stubble	Total
WL319HQ	-0.39	-0.50	-0.66	-0.52
WL343HQ	-0.45	-0.65	-0.78	-0.62
WL525HQ	-0.34	-0.59	-0.80	-0.57
Medoc Max	-0.52	-0.65	-0.78	-0.65
Zhongmu No.3	-0.32	-0.60	-0.75	-0.55
Baoding	-0.45	-0.63	-0.78	-0.62

Table 3

Synthetical allelopathic effect (SE) of methanol extracts from the first three stubbles of six alfalfa cultivars on barnyardgrass.
Variety	SE
Variety	First stubble	Second stubble	Third stubble	Total
WL319HQ	-0.30	-0.39	-0.37	-0.35
WL343HQ	-0.43	-0.29	-0.45	-0.39
WL525HQ	-0.33	-0.33	-0.40	-0.35
Medoc Max	-0.34	-0.36	-0.45	-0.38
Zhongmu No.3	-0.16	-0.28	-0.44	-0.29
Baoding	-0.33	-0.31	-0.51	-0.38

In the same way, the allelopathic effect of the extracts from Zhongmu No.3 on barnyardgrass was also lower than that of other varieties. Moreover, the SE value of the first stubble of alfalfa extracts from Zhongmu No.3 was − 0.16, which was also far weaker than the other two stubbles. Unlike green foxtail, there was almost no difference in the allelopathic effects of barnyardgrass among the three stubbles of four other varieties, especially between the first and second stubbles (Table 3).

The results in present research showed that the allelopathy of alfalfa was significantly different among cultivars, together with other published studies in alfalfa^{4, 9–11}. For example, the Q75 and Titan9 were the least allelopathic genotypes among the 40 alfalfa genotypes when the density was 15 lings/beaker ⁴. There were also significant differences in allelopathic effects of aqueous extracts from six different varieties of alfalfa seedling on orchardgrass (Dactylis glomerata)⁹. It was found that the allelopathic potential in alfalfa plants to lettuce varied with the eight common varieties in Japanese¹⁰. In addition, the inhibition effects of Phabulous, Longdong and Zhongmu No.1 on several weeds were stronger than that of other twelve tested cultivars¹¹. In addition, Zhang et al (2021) found that WL656HQ and 3105C were autotoxicity-tolerant and autotoxicity-sensitive varieties in twenty-two alfalfa varieties, respectively¹². As we all known, the phenolic compounds are important alfalfa allelochemicals, which are mainly derived from phenylpropane metabolism pathway, and phenylalanine ammonia-lyase (PAL) is the key enzyme of this metabolic pathway¹³. Li et al (2017) found that the activity of PAL and gene expression level of PAL genes in allelopathic rice (Oryza sativa) were higher than that in non-allelopathic rice or weakened allelopathic rice¹⁴. Therefore, the differences in weed inhibition among different alfalfa varieties provide potential genetic resources for the development of allelopathic alfalfa varieties. Further research is needed to determine which genes contribute to the differences in allelopathic ability among different alfalfa varieties. If corresponding allelopathic genes in alfalfa varieties can be combined with high-yield varieties, it will ultimately provide broad prospects for weed control.

There were significant differences in the allelopathic effects of alfalfa on weeds among different the times of harvests. Compared to the first and second stubbles of Zhongmu No.3, the third stubble showed the stronger inhibition on green foxtail and barnyardgrass, with corresponding SE values of -0.75 and − 0.44, respectively (Table 2–3). Chon et al (2006) found that the content of allelochemicals accumulated with the increase of planting years of alfalfa¹⁵. Various climate factors, for instance, temperature and precipitation might also play crucial roles in the release and degradation of allelochemicals¹⁶. The climate changes during the three cutting times may affect the accumulation of allelopathic substances in alfalfa. However, the allelopathic effects of extracts from some varieties on barnyardgrass did not increase with the increase of harvest times (Table 3), which was different from that of green foxtail (Table 2).

Both barnyardgrass and green foxtail were notorious weeds in worldwide, and which seriously restricted the yield and quality of alfalfa in northern China¹⁷. The results pointed to the possibility that the species-specific growth regulatory effects of allelochemicals. Compared with barnyardgrass, the corresponding green foxtail in this study was generally more susceptible to alfalfa extracts (Fig. 1–5). The reason for the sensitivity difference between the two weeds was not clear. In fact, the phenomenon of sensitivity difference to alfalfa extracts has also been reported in other weeds. For example, the seedlings of lambsquarters were the most susceptible to alfalfa extracts, and giant foxtail was the most resistant species among six weed species studied⁵. Although alfalfa has the potential as allelopathic weed control, its application in herbicide spectrum and other aspects still needs further research.

It was evident that that low concentrations of allelochemicals released by alfalfa may stimulate the growth of barnyardgrass or green foxtail. While if the allelochemicals exceed a certain dosage, the growth of weeds will also be inhibited. It is not difficult to find that the growth of root in the same weed was more inhibited than that of shoot (Fig. 2–5). The organ specificity of the inhibitory effect also occurs in other weeds such as annual ryegrass and crabgrass^{4, 18}. The result also corroborated earlier reports that the root growth was more susceptible to extracts than seed germination or hypocotyls growth³.

In this study, the allelopathic effects of six common alfalfa varieties on seed germination, seedling growth, root growth of green foxtail and barnyardgrass were comprehensively evaluated. We found that the 1) Medoc Max and Zhongmu No.3 exhibited the strongest and weakest allelopathic effects on the two types of weeds tested. 2) The inhibitory effect on green foxtail increases with the increase of harvest times for the same alfalfa variety. 3) The stronger inhibitory effect was discovered on green foxtail than that on barnyardgrass. 4) The growth of root in the same weed was more susceptible to the extracts than seedlings.

Preparation of extracts from aboveground parts of alfalfa. The six alfalfa varieties tested were WL319HQ, WL525HQ, WL343HQ, Medoc Max, Baoding and Zhongmu No.3, which were fully proved to be the main varieties suitable for planting in eastern Shandong. All the varieties were naked seed obtained by Beijing Zhengdao Seed Industry Co., Ltd, and the detailed information was listed in Table 1.

Table 1

Information of six alfalfa varieties tested.
Number	Variety	Fall dormancy	Source
1	WL319HQ	2.8	USA
2	WL343HQ	4.0	USA
3	WL525HQ	8.0	USA
4	Medoc Max	6.5	France
5	Zhongmu No.3	2.8	China
6	Baoding	-	China

The seeds of the above different alfalfa varieties were sown in the field of Qingdao (123.16°E, 37.49°N) on March 10, 2018. The aboveground part of alfalfa was cut on May 17th, June 20th, and July 15th of the year, respectively. A total of 500 g of the aboveground part of alfalfa was dried to constant weight at 37℃, stored at 4℃ after shearing. Accurate 25 g of the above sample was added into 500 mL of 70% methanol solution, and then was oscillated for 12 h at the speed of 150 rpm min^− 1 at 10℃. After 15 minutes of ultrasound, the mixture was repeatedly filtrated by vacuum with 0.45-µm membrane for three times. Subsequently, the collected filtrate was concentrated into the paste by rotary evaporation at 40℃ and 50 rpm min^− 1. Dissolved the above paste with deionized water and diluted it to 10 mL to obtain an extraction solution with a final concentration of 2.5 g mL^− 1.

Sterilization of seeds of green foxtail and barnyardgrass. The seeds of green foxtail and barnyardgrass in the research were collected from the alfalfa planting area. The collected weed seeds were washed for three times, and then sterilized with 75% alcohol for one minute. After cleaning with sterile water for three times, immerse the seeds in 30% NaClO for 15 minutes, and then clean with sterile water again.

Effects of alfalfa extracts on seed germination, seedling growth, and root growth of green foxtail and barnyardgrass. The treatment solutions with different concentrations (0.01, 0.03, 0.05 and 0.07 g mL^− 1) were prepared by diluting the above extraction solution with sterile water. A total of 50 sterile weed seeds were evenly placed in a petri dish, and then 5 mL of extract solution with corresponding concentration was added. The control group was treated with equal volume of sterile water. All the dishes were kept in artificial climate chambers with a regime temperature of 25/20°C (light/dark) and a 16-h photoperiod with a luminous intensity of approximately 15 000 lx. The experiments were conducted with three replicates per dose.

The seed that produced at least 1 mm radicle was considered as germinated. After 8 days of cultivation, the germination rate of barnyardgrass seeds was determined. Similarly, the determination time of green foxtail was 10 days after treatment. At the same time, 10 seedlings were randomly selected from each petri dish, and the length and fresh weight of seedlings and roots were measured respectively.

Statistical analysis. Statistical analysis was performed using SPSS software (v. 20.0, IBM, Armonk, NY, USA), and the data are expressed as mean ± standard deviation. The Dunnett’s test at the 5% level of significance in one-way analysis of variance (ANOVA) was carried out to compare the allelopathy under different treatments.

The response index (RI) of alfalfa extracts to weeds is calculated according to the following formula. In this equal, C and T represent the values of control group and treatment group respectively. If the RI value is positive, the effect of the extracts on weeds is promoted. On the contrary, it is inhibition. The absolute value of RI reflected the magnitude of the allelopathy.

RI = (T-C)/T (T ≥ C)

RI = (T-C)/C (T<C)

The SE represents the arithmetic mean of the RI of five measurement index of weeds, including germination rate, the length and fresh weight of seedings, the length and fresh weight of roots¹⁹. In addition, the values of SE on green foxtail and barnyardgrass for each alfalfa varieties with three different stubbles were also calculated respectively.

Competing interests

The authors declare no conflict of interest.

Author Contribution

X.Y.F., G.X.H. and S.J. designed the research and performed all analyses. X.F.R wrote the first draft of the manuscript. B.Q., C.S.P. and M.F.H. implemented the experiment and helped to collect data. L.Q.W. provided material. All authors reviewed the manuscript.

Acknowledgements

This work was supported by the China Agricultural Research System (CARS-34), National Natural Science Foundation of China (32302407) and Doctoral Scientific Research Startup of Qingdao Agricultural University (6631123002).

Data Availability

All data generated or analysed during this study are included in this article.

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No competing interests reported.

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Allelopathic interference of six alfalfa varieties at different harvesting stages on the germination, seedling and root growth of green foxtail and barnyardgrass

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Abstract

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Introduction

Results

Discussion

Methods

RI = (T-C)/T (T ≥ C)

RI = (T-C)/C (T<C)

Declarations

Competing interests

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

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