Allelopathy of Wheat and Faba Bean Extracts in an Intercropping System

We intensively studied faba bean (Vicia faba L.) and wheat (Triticum aestivum L.) intercropping and found that this type of intercropping can effectively control the occurrence of faba bean wilt under eld conditions. We conducted hydroponic experiments to explore the role of plant extracts in process of soilborne diseases and the mechanism of disease control of faba bean and wheat intercropping. In this experiment, three concentration gradients of faba bean and wheat stems and leaves and root extracts were added to study the effects of faba bean and wheat extracts on faba bean growth, root physiological resistance and the growth of Fusarium oxysporum f. sp. fabae (FOF).


Result
Faba bean extracts signi cantly inhibited the growth of faba bean seedlings and the activity of root defense enzymes and signi cantly stimulated the growth of FOF at high concentrations. Compared with the treatment with faba bean extracts, wheat extracts signi cantly enhanced the growth of faba bean seedlings, increased the activity of defense enzymes and inhibited the growth of FOF.

Conclusions
Based on these results, we believe that wheat extract can effectively alleviate the autotoxicity of faba beans and also control the occurrence of faba bean wilt in the eld. This provides a theoretical basis for practical intercropping to reduce the harm of faba bean wilt.

Background
Continuous planting and harvest of single crops is a common practice in modern agriculture, and that has resulted in serious obstacles. The hazards of continuous cropping obstacles primarily include soil compaction, the frequent occurrence of soil-borne diseases, a reduction in crop yields or even a total lack of germination of the seeds. Among them, the frequent occurrence of soil-borne diseases has always been a very di cult problem during actual production (Young, C.C., 1984; Grodzinsky, A.M., 1992). Thus far, soil-borne diseases have seriously threatened the production of various cash crops, such as watermelon, peanut, and cotton, which has a substantial impact on agricultural production around the Other studies have shown that the main reason that autotoxic substances can promote the occurrence of diseases is that they can have a strong destructive effect on plant physiological and biochemical resistance. For example, Ye SF et al found that cinnamic acid in cucumber autotoxic substances destroyed the plant antioxidant system, increased the content of active oxygen free radicals in the root and accelerated the degree of membrane lipid peroxidation, thereby rendering the plants more susceptible to infection and increasing the incidence of disease. Several chemical and biological methods have been developed to control plant diseases (Gil VS et.al, 2008). However, these methods are not environmentally friendly or su ciently e cient (Minuto A et al, 2006;Li, X.et al, 2018).
Intercropping is a planting method that planting two or more crops in close proximity (Li, X.et al, 2018). In actual production, it is used as a green and e cient planting method to control soil-borne diseases and increase the yields of crops (Ren, L et al,2016Li, X. G,2014. Allelopathy is an indispensable part of the study of the disease control mechanism of intercropping. For example, in the wheat/watermelon intercropping system, wheat allelopathic substances secreted by the root system increase the expression of watermelon defense genes, improve the ability of watermelon to resist the invasion of pathogen and control the occurrence of wilt (Huifang, L et al 2018). In the intercropping system of cumin (Cuminum cyminum L.) and watermelon, cumin acid secreted by the root system of C. cyminum signi cantly increased the activity of antioxidant enzymes and defensive enzymes in the watermelon roots and improved the ability of watermelon to resist pathogen (Sun, Y et al,2017). Allelopathic chemicals can enter the environment in different manners to play a role in the direct or indirect effects on growth of plants. The primary manners in which allelopathic chemicals are released include following their release by aboveground volatilization and leaching, secretion by roots and the decay of stubble (Guo, K et al 2016). Root secretion is the main source of allelochemicals belowground; these substances enter the soil directly through secretions from the plant roots and play a role (Ren, L et  However, because of the continuous single planting, the yield of faba beans is greatly reduced owing to Fusarium wilt (Stoddard et al, 2010). In Yunnan and southwestern China, faba beans are usually planted with wheat to control faba bean wilt. We intensively studied the mechanism of control of faba bean and wheat intercropping to control the wilt disease of faba bean. We explained the mechanism of control of faba bean and wheat intercropping from the aspect of biodiversity. However, there is a lack of data on the ability of plant extracts to cause disease. We conducted a preliminary experiment on the allelopathy of extract of faba bean stems and leaves based on a eld experiment but using hydroponics. In this study, we aimed to (i) reveal the allelopathic capability of extracts from faba beans and wheat; (ii) explore the mechanism of disease control from a wheat and faba bean intercropping system and lay the groundwork for further research; and (iii) provide effective theoretical guidance for actual agricultural production to achieve the most effective disease control.

Results
Effect of intercropping wheat and faba bean on Fusarium wilt of faba bean Figure 2A shows that the incidence of faba bean wilt during the mature and owering periods was signi cantly higher than that during the branching period in the monocropping and intercropping models.
Compared with monocropping, intercropping wheat and faba bean signi cantly reduced the incidence of faba bean wilt in the branching and owering stages by 20.63% and 13.36%., respectively In Fig. 2B, the disease index of faba bean wilt during the owering stage is signi cantly higher than that during the branching stage, and the disease index in mature stage of faba bean wilt is signi cantly higher than that in the owering stage. The disease index gradually increases with time. Compared with monocropping, intercropping wheat and faba bean signi cantly reduced the disease index of faba bean wilt by 51.64%, 37.78% and 29.72% during the branching stage, owering stage and mature stage, respectively. Figure 2AB shows that intercropping with faba bean and wheat can effectively control the faba bean wilt compared with the faba bean monocropping, and the effect is particularly signi cant in the suppression of faba bean wilt disease index. The branching period is the period when the faba bean and wheat intercropping is the most effective at controlling disease. The incidence of faba bean wilt and the disease index decreased by 20.63% and 51.64% respectively.
Effects of wheat and faba bean stem, leaf and root extracts on faba bean growth As shown in Table 1A, compared with the control, the addition of three concentrations of faba bean stem and leaf extracts signi cantly inhibited the growth index of faba beans and was concentration dependent. Compared with the control, the exogenous addition of 0.01g·mL − 1 wheat stem and leaf extracts signi cantly increased the plant height, dry weight and root length of faba bean. Exogenously added 0.05 g·mL − 1 wheat stem and leaf extract slightly increased these parameters compared with the control. However, when the concentration of wheat stem and leaf extract reached 0.1 g·mL − 1 , it signi cantly inhibited all the growth indices of faba bean (Fig. 3). Table 1B shows that, compared with the control, the addition of 0.01 g·mL − 1 faba bean root extract signi cantly inhibited the main root length and root length of faba bean but had no signi cant effect on the other indicators. Compared with the control, the faba bean root extract with a concentration greater than or equal to 0.05 g·mL − 1 signi cantly inhibited all the growth indices of faba bean. In contrast, wheat root extract had the opposite effect. Compared with the control, the addition of 0.01 g·mL − 1 wheat extract signi cantly increased all the growth indices of faba bean with the exception of number of leaves. The addition of 0.05 g·mL − 1 wheat root extract signi cantly increased the main root length, stem dry weight, root dry weight and root length of faba bean. However, when the concentration of wheat root extract reached 0.1 g·mL − 1 , it signi cantly inhibited the plant height, main root length, stem weight, and root length of faba bean compared with the control and had no signi cant effect on the other indicators ( Fig. 3).
The most notable effect is that the wheat extracts signi cantly increased the growth index of faba beans at three concentrations compared with the faba bean extracts. The data is an average and the standard error of three repetitions is represented by a bar. Different letters in the gure indicate signi cant differences between different treatments (P < 0.05). The data is an average, and the standard error of three repetitions is represented by a bar. Different letters in the gure indicate signi cant differences between different treatments (P < 0.05).
Effects of extracts from faba bean stems, leaves and roots on the physiological resistance of faba bean roots A shown in Fig. 4A, compared with the control, the addition of 0.05 and 0.1 g·mL − 1 faba bean stem and leaf extracts signi cantly reduced the POD activity of the faba bean root system. Compared with the faba bean stem and leaf extracts, the wheat stem and leaf extracts signi cantly increased the POD activity of the faba bean root in all concentrations tested. Figure 4B shows that the faba bean root extracts signi cantly reduced the POD activity of faba bean root in the 0.1 g·mL − 1 treatment compared with that of the control. Compared with the faba bean root extracts, the wheat root extracts can signi cantly increase the POD activity of the faba bean root at all concentrations tested. Figure 5A reveals that compared with the control, the faba bean stem and leaf extracts signi cantly inhibited the activity of CAT in the faba bean root system at 0.05 and 0.1 g·mL − 1 concentrations. Compared with the faba bean stem and leaf extract, the wheat stem and leaf extracts can signi cantly increase the activity of CAT in the faba bean root system at all concentrations tested. As shown in  (Fig. 7A). Figure 7B shows that the faba bean root extracts signi cantly . In this experiment, the extracts of faba bean stems, leaves, and roots at low concentrations inhibited the spore germination and mycelial growth of FOF. However, with the increase in concentration, the inhibitory effect gradually disappeared, and at high concentration, the extracts signi cantly promoted germination and growth of the fungus. In actual agricultural production, owing to years of continuous cropping, the faba bean extracts had accumulated to large amounts in the soil, and the concentration of extract in the soil is very high. Therefore, in actual production, the faba bean extract has an enormous stimulatory effect on the germination and growth of FOF. Based on these results, we hypothesize that the autotoxicity of faba bean may promote the growth of pathogen by destroying the defense system of faba bean root system and enhancing the invasion of pathogen to the root system of faba bean, nally resulting in strong inhibition of the growth of faba beans.
Intercropping is a green and e cient planting model, particularly in terms of increasing yield and controlling diseases. Now this advantage has been veri ed in many intercropping systems, such as corn and soybean intercropping that effectively controls corn crown rot and rice/watermelon intercropping that effectively controls watermelon wilt (Gao, X et al,, 2014; Ren LX et al, 2008). Similarly, we found that in eld experiments, intercropping faba bean and wheat signi cantly inhibited the incidence of faba bean wilt in the faba bean branching and owering stages, and during the branching, owering and podding stages of faba bean, the disease index of faba bean wilt was signi cantly inhibited. Most research on the mechanism of intercropping disease control focuses on allelopathic substances secreted into the soil through the root system, but in actual production, these compounds can also enter the soil through the leaching and evaporation of plant roots, stems and leaves. These allelopathic substances are easily overlooked (Hao, Z. P et al, 2007). For the research on the mechanism of control by faba bean and wheat intercropping, we added different concentrations of wheat stem and leaf and root extracts in the faba bean hydroponic experiment. Compared with the faba bean extracts, we found that the wheat extracts signi cantly promoted the growth of faba bean seedlings at all treatment concentrations; similar conclusions have been obtained in the moringa and wheat intercropping system (Khan et al, 2017). We also simultaneously found that, compared with the treatment of faba bean extracts, wheat extracts signi cantly enhanced the activities of faba bean root POD and CAT and effectively reduced the accumulation of faba bean root MDA. The ability of the faba bean root system to resist the invasion of pathogen had improved. A series of results show that in actual production, wheat extracts can effectively alleviate the autotoxicity of faba beans. We hypothesize that this may be one of the important mechanisms of wheat and faba bean intercropping for disease control. This result is consistent with previous studies on rice/watermelon, rice/water chestnut, and corn/sun ower intercropping systems (Ren et al, 2008;Chen et al, 2012;Qin et al, 2013). On the basis of the signi cant improvement of the faba bean root defense system by wheat extracts, compared with the faba bean extracts, the wheat extracts could signi cantly inhibit the mycelial growth and germination of spores, thereby fundamentally reducing the possibility of pathogen infection of faba beans. This is consistent with the results of Ren et al (2010) on the wheat/watermelon intercropping system. This shows that in the wheat/faba bean intercropping system, the extracts of wheat can effectively relieve the stimulatory effects of the faba bean extracts on the occurrence of faba bean wilt, thereby further reducing the occurrence of faba bean wilt. Unexpectedly, compared with the control, the wheat extracts were effective at a low concentration, but they enhanced the inhibition of the growth of faba beans at high concentration. However, in actual production, unlike the large accumulation of faba bean extract, wheat has no continuous cropping history. The concentration of extract in the eld is very low and is easily degraded by microorganisms in the soil. Therefore, in the actual eld intercropping mode, the concentration of wheat extracts will not be very high. This experiment was a hydroponic one, and our aim was to explore the allelopathy of wheat extracts of different concentrations. This does not examine the decomposition of allelochemicals by soil rhizosphere microorganisms. However, it also reminds us that in actual agricultural production, we should pay attention to controlling the ratio of faba bean and wheat and avoiding an excessive planting density of wheat that leads to an excessive concentration of rhizosphere wheat extract that could inhibit the growth of faba bean. After veri cation, we believe that the 2:6 ratio of faba bean:wheat used in this experiment is the best ratio to inhibit FOF, which is of substantial signi cance for the guidance of eld production and disease control.

Conclusions
In summary, wheat/faba bean intercropping can effectively control the occurrence of faba bean wilt.
Studies on the extracts of faba beans and wheat found that the extracts of wheat improved the condition of faba bean seedlings, enhanced the physiological resistance of faba beans, eased the autotoxicity of faba beans and suppressed pathogenic fungal growth. All of these actions can reduce the occurrence of faba bean wilt under eld conditions. This experiment should effectively guide the cultivation of faba bean and wheat intercropping in actual production. It can maximize the ability of faba bean and wheat intercropping to control disease and is highly signi cant to actual agricultural production. Although this is only preliminary research, it provides encouraging results and a basis for future research.
FOF was isolated from continuously cropped faba beans elds by the Plant-Microbe Laboratory at Yunnan Agricultural University, China. The fungus was transferred to potato dextrose agar (PDA) media, incubated at 28 °C for 7 days, and then stored at 4ºC.

Field Trials
The eld test was conducted in the experimental eld of Hanbao, Kunming, Yunnan Province, China from October 2018 to May 2019. There was moderate rainfall during planting. The eld lies in the humid subtropical zone and has a paddy soil type with topsoil (0 ~ 20 cm) that contained organic matter 14.5 g/kg, total nitrogen 1.21 g/kg, alkali nitrogen 59.8 mg/kg, available phosphorus 29.9 mg/kg, available potassium 52.1 mg/kg and had a pH of 6.5.
The faba beans were monocropped (MF) or intercropped with wheat (IF) in plots that measured 5.4 m × 6 m with a total area of 32.4 m 2 . As shown in Fig. 1, the MF faba bean plants were sown at 0.1 m intervals, and the rows were spaced 0.3 m apart. Six rows of wheat and two rows of faba beans were planted alternately in the IF plot for a total of three and four strips, respectively. The faba bean rows and intercropping faba bean and wheat rows were each spaced 0.3 m, whereas the wheat rows were spaced 0.2 m. The faba bean plants from the outermost rows of the 1st and 4th strips were not sampled. In addition, a 1 m-wide faba bean strip was planted around the entire test eld as a protection line. Each treatment was repeated three times in six random blocks. No pesticides, fungicides or herbicides were applied throughout the growth period. Other management was conducted according to the local agronomic customs.

Measurement Of The Incidence Of Fusarium Wilt
The severity of the disease was scored at different stages as: 0 -no symptoms of infection; 1 -slight plaques or discoloration at the base of the stem or peripheral roots; 2 -uneven lesions at the base of root or stem; 3 -uniform lesions, discoloration or wilting in 1/3 to 1/2 of the stem base or root and a reduction in lateral roots; 4 -completely discolored or withered roots or stem base, and 5 -complete wilting of the plant and death. The disease index and wilt incidence were calculated as:

Preparation Of The Aqueous Extracts
At maturity, all the faba bean and wheat plants were collected from the experimental eld, and the dust that adhered to the plant and root systems was rinsed with tap water and then deionized water. The plants were divided into two parts, roots and a combination of stems and leaves, which were desiccated in an oven at 105 °C for 30 min, dried at 65 °C to a constant weight and cut into small pieces 1 cm long.
Twenty gram of dry samples of the roots, stems and leaves were weighed, respectively. A volume of 200 mL of deionized water was added and shaken at a constant temperature for 2 h. The solution was ltered through three layers of gauze after leaching at room temperature for 48 h and centrifuged at 4000 r·min − 1 for 10 min. The supernatant was considered to be 0.1g·mL − 1 plant water infusion mother liquor and stored at -20℃ for use.

Greenhouse cultivation
Faba bean seeds were soaked for 24 h at room temperature, germinated at 25 °C and sown in sterile quartz sand that had been soaked in Hoagland nutrient solution. Once the faba bean seedlings had grown 4-6 leaves, they were transplanted into 2 L containers that contained various concentrations of aqueous extracts. The treatments included 0 (control), 0.01, 0.05 and 0. minute per gram of fresh weight sample (mg · g − 1 · min − 1 ).
To measure the content of malondialdehyde (MDA), the end product of membrane lipid peroxidation (Bird BR, 1983), 0.5 g of the plant sample was homogenized in 5 ml of 5% trichloroacetic acid and centrifuged at 3000 rpm for 10 min. The supernatant was aspirated, and 2 ml was boiled with the same volume of 0.67% thiobarbituric acid for 30 min, cooled and centrifuged. The absorbance was measured at 450, 532 and 600 nm.

Evaluation Of Fof Growth And Conidial Germination
Mycelial discs that were 9 mm in diameter were plated onto PDA and cultivated at 28 °C for 7 days. The colony diameter was measured radially in three directions on days 3 and 7. A 9 mm agar plug was cut from the 7-day-old culture, inoculated into 15 ml PD media containing 0, 0.01, 0.05 or 0.1 g·mL − 1 faba bean or wheat aqueous extracts, and incubated for 7 days at 28℃ with constant shaking at 170 rpm.
The culture broth was ltered, dried at 80 °C for 12 h and weighed to determine the fungal biomass. The germination of spores was determined by washing the 7-day-old mycelia on PDA with sterile water and collecting the spores by ltration through four layers of gauze. The spore suspension was diluted to ≤ 1 × 10 3 CFU/ml, and 0.1 ml of spores were plated on each 2% (w/v) water agar plate containing 0, 0.01, 0.05 or 0.1 g·mL − 1 faba bean or wheat aqueous extracts. The plates were incubated at 28℃ for 3 days, and the number of colonies was counted. Each experiment was repeated in triplicate.

Statistical analysis
The allelopathic effect of the extract was measured according to the response index (RI) proposed by Williamson and Richardson (1988).    Effect of extracts from faba bean stems, leaves and roots on POD activity of faba bean roots.BR: treatment with exogenously added faba bean root extract; BSY: treatment with exogenously added faba bean stem and leaf extract; WR: treatment with exogenously added wheat root extract; WSY: treatment with exogenously added wheat stem and leaf extract. The data is an average, and the standard error of three repetitions is represented by a bar. Different letters in the gure indicate signi cant differences between different treatments (P<0.05).

Figure 5
The effects of extracts from faba bean stems, leaves and roots on CAT activity of faba bean roots.BR: treatment with exogenously added faba bean root extract; BSY: treatment with exogenously added faba bean stem and leaf extract; WR: treatment with exogenously added wheat root extract; WSY: treatment with exogenously added wheat. The data is an average, and the standard error of three repetitions is represented by a bar. Different letters in the gure indicate signi cant differences between different treatments (P<0.05).

Figure 6
Effects of extracts from faba bean stems, leaves and roots on the MDA content of faba bean roots.BR: treatment with exogenously added faba bean root extract; BSY: treatment with exogenously added faba bean stem and leaf extract; WR: treatment with exogenously added wheat root extract; WSY: treatment with exogenously added wheat stem and leaf extract. The data is an average, and the standard error of three repetitions is represented by a bar. Different letters in the gure indicate signi cant differences between different treatments (P<0.05).