Preventive effect of Cleome spinosa against cucumber Fusarium wilt and improvement on cucumber growth and physiology

Abstract

Cucumber wilt is an important soil borne disease in cucumber production, which seriously affects the development of the cucumber industry. Cleome spinosa also has pharmacological effects such as antibacterial, analgesic, anti-inflammatory, and insect repellent. To study the control effect and mechanism of Cleome spinosa fumigation on cucumber wilt disease, different concentrations of Cleome spinosa fragments were applied on cucumber plants infected with Fusarium oxysporum. Cleome spinosa fumigation significantly reduced the incidence rate of cucumber Fusarium wilt. Under the fumigation treatment of 7.5 g kg⁻¹ Cleome spinosa fragments, the preventive effects were 74.7%. Cleome spinosa fragments fumigation can promote cucumber growth and synthesis of photosynthetic pigments, thereby improving individual plant yield and fruit quality. At 7.5 g kg⁻¹ Cleome spinosa fragments fumigation treatment, the plant height and individual plant yield of cucumber increased by 20.3% and 34.3%, respectively. Cleome spinosa fumigation can enhance the activity of antioxidant enzymes in cucumber, maintain a balance of reactive oxygen species metabolism, and enhance the plant disease resistance. Moreover, Cleome spinosa can also regulate the activities of Mg²⁺-ATPase and Ca²⁺-ATPase, enhancing its resistance to Fusarium oxysporum. Moreover, number of bacteria and fungi significantly decreased under Cleome spinosa fumigation. Those results suggested that Cleome spinosa could effectively restrain cucumber Fusarium wilt. This study will provide a new idea for the further use of biological fumigation to prevent soil-borne diseases.

Introduction

Cucumber (Cucumis sativus L.) represents a vital vegetable crop in the world, and with the continuous development of horticultural facilities, the planting acreage of cucumber has also increased (Liu et al. 2021). Numerous abiotic and biotic variables lead to the low cucumber production, and amongst the biotic factors, Fusarium wilt is the most prominent and restricts the yield of cucumber in all regions worldwide (Naguib et al. 2021). Agricultural, physical, biological and chemical treatments are the main measures of controlling soil-borne diseases. Among them, the use of chemical agents in soil disinfection and fumigation is effective for the prevention and control of cucumber Fusarium wilt; however, chemical agents not only pollute the soil but also cause great harm to human health (El-Sharkawy et al. 2016). Therefore, a biological fumigant must be looked into that can be used as a substitute for chemical agents to pose no risk to the environment, humans and animals.

In recent years, biofumigation has become a popular and environmentally friendly controlling strategy against Fusarium wilt (Guo et al. 2018). Biofumigation can effectively control some soilborne plant pathogens, reduce the use of chemical pesticides, increase crop yields, effectively improve the soil structure and increase the content of organic matter and beneficial microorganisms (Wang et al. 2020).

Cleome spinosa Jacq. (Capparaceae) is a herbaceous plant that is native to tropical regions in South America (Bezerra et al. 2019). In addition to its ornamental value, Cleome spinosa also has pharmacological effects such as antibacterial, analgesic, anti-inflammatory, and insect repellent. It was shown in our previous study that Cleome spinosa had antibacterial activity against Fusarium oxysporum (Zhang et al. 2021). However, there have been few research reports on the application of Cleome spinosa to control soil borne diseases of plants. This study aimed to explore the preventive effect of Cleome spinosa against cucumber Fusarium wilt, and its improvement on cucumber growth and physiology. This study will provide a new idea for the further use of biological fumigation to prevent soil-borne diseases.

Materials and methods

Climatic conditions of the experimental field

The research site is in the north temperate zone and continental monsoon area (rainy and hot during the summer; cold and arid during the winter) of China that receives an average annual precipitation of 480.8 and 651.2 mm in 2017 and 2018, respectively.

Plant materials and tested strains

The cucumber line 649 is susceptible to the pathogen F. oxysporum. Seeds of this line were obtained from the Cucumber Laboratory of Northeast Agricultural University. Isolate H28 of F. oxysporum f. spp. Cucumerinum was supplied by the Plant Pathology Laboratory of Northeast Agricultural University, China.

Experimental design

The experiment was conducted at Northeast Agricultural University (126° 63′ E and 45°44′ N; Harbin, China) in 2017 and 2018. The experimental soil was derived from the cultivation layer (0–20 cm) of an experimental field of the University. Cleome spinosa fragments soil were mixed in pre-determined proportions (mass ratios). Eight treatments were prepared: CK, the control without any treatments; Z0, vaccinations with Fusarium oxysporum; Z1, 1.5 g kg⁻¹ Cleome spinosa fragments + vaccinations with Fusarium oxysporum; Z2, 3.0 g kg⁻¹ Cleome spinosa fragments + vaccinations with Fusarium oxysporum; Z3, 4.5 g kg⁻¹ Cleome spinosa fragments + vaccinations with Fusarium oxysporum; Z4, 6.0 g kg⁻¹ Cleome spinosa fragments + vaccinations with Fusarium oxysporum; Z5, 7.5 g kg⁻¹ Cleome spinosa fragments + vaccinations with Fusarium oxysporum; and L, 6.0 g kg⁻¹ chloropicrin + vaccinations with Fusarium oxysporum. Each experimental plot had an area of 1800 cm², and three biological replicates were used. The soil was treated and fully mixed with the pre-determined proportions of Cleome spinosa fragments by the fertilization method, fully-watered, and covered with film for 14 days. Then cucumber seedlings were transplanted into the pot soil in different treatments in late May.

Disease incidence and preventive effect of Cleome spinosa

Isolate H28 of F. oxysporum was incubated in liquid potato-sucrose medium with shaking (110 rpm) at 25 °C for 7 days. Spores were filtered through a double layer of cheesecloth, and the suspension was centrifuged at 4000×g for 5 min at 4 °C. The spores were then resuspended in distilled water, and the concentration was adjusted to 1 × 10⁷ spores mL⁻¹.

Isolate H28 was inoculated by pouring the spore suspension on the roots (Zhou et al. 2010). One hundred milliliters of F. oxysporum per plant was inoculated via root irrigation 7 days after transplantation. Disease occurrence was investigated at 14 day after inoculation, and the degree of disease occurrence was recorded according to previously described standards for disease levels (Singh et al. 1999): 0, no disease characteristics; (1) mild disease characteristics in hypocotyl or cotyledon with no effect on growth; (2) obvious necrotic spots on hypocotyl or cotyledons, or yellow leaves, affecting normal growth; (3) two cotyledons turning yellow, or one withers; (4) two cotyledons growth stiffening, and some of them wilting; (5) wilting of whole plants, even lodging or withering.

$$\text{Disease}\text{index}=100\times \sum \left(\text{Number}\text{of}\text{diseased}\text{plants}\text{per}\text{grade}\times \text{Representative}\text{value}\text{of}\text{each}\text{level}\right)/\left(\text{Total}\text{number}\text{of}\text{plants}\times \text{Top}\text{representative}\text{value}\right),$$

$$\text{Disease}\text{preventive}\text{effect}\left(\%\right)=\left(\text{Disease}\text{index}\text{of}\text{control}\text{area}-\text{Disease}\text{index}\text{of}\text{treatment}\text{area}\right)/\text{Disease}\text{index}\text{of}\text{control}\text{area}\times 100.$$

Investigation of plant growth indices and photosynthetic pigments contents

The plant height, stem diameter and dry matter accumulation of 10 cucumber plants from each of three biological replicates were measured during the full-fruit period. The contents of photosynthetic pigments were determined using the ethanol acetone method (Zhang et al. 2009). Photosynthetic pigments were extracted from fresh samples in 80% acetone. Each extract was centrifuged at 3000×g for 5 min. The absorbance of the supernatant was measured at 470, 645, and 663 nm with a UV-754 spectrophotometer (Zealquest Scientific, Shanghai, China). Contents of Chlorophyll (Chl) a, Chl b, and Carotenoids (Car) were calculated using adjusted extinction coefficients. Pigment contents were expressed as mg g⁻¹ (fresh mass, FM).

Determination of cucumber fruit quality and yield per plant

Vitamin C, free amino acid, soluble sugar, soluble protein and nitrate contents were measured by 2,6-dichlorophen-based colorimetry, ninhydrin staining, anthrone-based colorimetry, Coomassie brilliant blue G-250 staining and salicylic acid-based colorimetry, respectively (Zhang et al. 2009). The weight of each cucumber fruit from each plant was measured using an electronic balance, and the yield per plant was calculated.

Determination of cucumber physiological indexes

SOD activity, POD activity, and CAT activity were measured in accordance with the nitro blue tetrazolium (NBT) photoreduction method, guaiacol method and ultraviolet absorption method, respectively (Zhang et al. 2009).

The activities of hexokinase and pyruvate kinase were measured according to the method of Schaffer and Petreikov (1997). Succinate dehydrogenase and malic dehydrogenase activities were determined using the method of Jenner (2001). The activities of Mg²⁺-ATP and Ca²⁺-ATP enzymes were in accordance with the method of (Zhang et al. 2009).

Determination of number of rhizosphere microorganisms

The degree of influence of changes in microbial levels in soil treated with Cleome spinosa was measured using plate dilution counting method. The soil was prepared as a suspension and subjected to gradient dilution. An appropriate dilution ratio was selected, and 100 µL of drops were added to the solid plate of the culture dish. After uniform coating, the levels of aerobic microorganisms (fungi and bacteria) in the colony and one gram of soil were calculated. Dry soil of 10.0 g was weighed, and transferred to a conical flask (250 mL), and 90 mL of sterilized distilled water was added to obtain a 10^−onefold dilution solution, mixed thoroughly for 10 min to obtain a suspension, then 1 mL were taken from it and placed in a test tube containing 9 mL of sterilized distilled water in advance. After mixing, a dilution solution with a concentration of 1 × 10⁻² was obtained. After continuous dilution, the final diluent concentrations obtained are 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, and 10⁻⁷, respectively.

The selected culture media for the determination of bacteria and fungi in soil are beef extract peptone agar (NA) and potato sucrose agar (PSA). Before use, a 25% lactic acid solution was added to the melted PSA medium to inhibit bacterial growth, and 3–4 drops of 25% lactic acid were added to every 100 mL of medium. When it is about to solidify, pour about 15 mL of liquid culture medium into it, add 0.5 mL of sample diluent to each culture dish, and invert it in a constant temperature incubator (25 °C) for 3 days. After cultivation, observe and count the number of colonies. Record the number of fungi for 5 days and calculate the microbial content in the soil sample based on various statistical results. When it is about to solidify, about 15 mL of liquid culture medium was poured into it, 0.5 mL of sample diluent was added to each culture dish, and inverted in a constant temperature incubator (25 °C) for 3 days. After cultivation, the number of colonies were observed and counted. The number of fungi was recorded for 5 days and the microbial content in the soil sample was calculated based on various statistical results.

The basic unit of microbial count is CFU g⁻¹. The number of bacteria in one gram of sample = the average number of colonies repeated several times at the same dilution level × five × Dilution factor. The repetition coefficient of each dilution plate is 3.

Results

Preventive effect of Cleome spinosa against cucumber Fusarium wilt

Cucumber seedlings were severely affected by Fusarium wilt and showed better growth with the increase of Cleome spinosa dosage (Fig. 1A). Disease indexes significantly decreased under different dosages of Cleome spinosa fumigation treatments (Fig. 1B). The preventive effects of Cleome spinosa fumigation gradually rose along with its increasing dosage. The value of disease index declined to the minimum (23) and that of preventive effect elevated to the maximum (74.7%) at Z5 treatment. When the dosage of Cleome spinosa reached 6.0 g kg⁻¹, its preventive effect no longer significantly rose. Moreover, the preventive effects of Z4 and Z5 treatments were slightly better than that of the L treatment.

Fig. 1.

Effects of Cleome spinosa fumigation on a morphology and b disease index of cucumber infected with F. oxysporum. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

Effects of Cleome spinosa fumigation on growth of cucumber seedlings

All the growth indices of cucumber seedlings gradually elevated with an increase in Cleome spinosa fumigation dose (Fig. 2). Under the Z5 treatment, plant height, stem diameter, shoot dry mass and root dry mass significantly increased by 20.3%, 17.5%, 21.2% and 20.0% compared to those under the control, respectively, and reached the peak values 55.7 cm, 0.47 cm, 6.3 g and 0.24 g, respectively.

Fig. 2.

Effect of different doses of Cleome spinosa fumigation on cucumber growth in the seeding stage. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

Effects of Cleome spinosa fumigation on yield and fruit quality of cucumber

All the fruit quality indices and yield of cucumber steadily elevated with the increasing Cleome spinosa fumigation dosage (Fig. 3). At Z5 treatment, the contents of vitamin C, free amino acid, soluble protein and nitrate in cucumber fruit, and yield per plant of cucumber significantly increased by 14.3%, 11.9%, 23.2%, 23.4% and 34.3% compared with those of the control, respectively, and reached the maximum values 0.352 mg g⁻¹, 0.488 mg g⁻¹, 2.271 mg g⁻¹, 0.274 mg g⁻¹ and 1.802 kg, respectively. There was no significant difference in soluble sugar content of cucumber fruits among different treatments.

Fig. 3.

Effect of different doses of Cleome spinosa fumigation on yield and fruit quality of cucumber. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

Effects of Cleome spinosa fumigation on photosynthetic pigments contents of cucumber

Photosynthetic pigments contents continuously rose with the increase of Cleome spinosa dosage (Fig. 4). Chl a and Chl b contents significantly increased by 11.2% and 22.9% under the Z5 treatment compared to those of the control, respectively, and reached the peak values 11.9 and 4.3 mg g⁻¹, respectively. There was no significant difference in Car content in cucumber leaves between different treatments.

Fig. 4.

Effect of different doses of Cleome spinosa fumigation on photosynthetic pigments contents in leaves of cucumber plant. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

Effects of Cleome spinosa fumigation on antioxidase activities of cucumber

With the increase in the fumigation dose of Cleome spinosa fragments, the CAT enzyme activity of cucumber leaves first gradually increased, and when the fumigation dose reached 6.0 g kg⁻¹, the CAT activity no longer increased, even decreased (Fig. 5A). The CAT enzyme activity in cucumber leaves at Z1, Z2, Z3, Z4 and Z5 was significantly higher than that of the control at seedling, flowering, and fruit stages. At Z4 treatment, the CAT activity in cucumber leaves increased by 333.3%, 304.5%, and 189.3% compared to the control at seedling, flowering, and fruit stages, respectively. Under the control treatment, the CAT enzyme activities of cucumber leaves were 18 U g⁻¹ min⁻¹, 22 U g⁻¹ min⁻¹, and 28 U g⁻¹ min⁻¹ at seedling, flowering, and fruit stages, respectively; whereas the CAT activity at three stages was 78 U g⁻¹ min⁻¹, 89 U g⁻¹ min⁻¹ and 81 U g⁻¹ min⁻¹ at seedling, flowering, and fruit stages, respectively, under the Z4 treatment.

Fig. 5.

Effect of different doses of Cleome spinosa fumigation on antioxidase activities of cucumber. a CAT activity. b SOD activity. c POD activity. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

The SOD activity in cucumber leaves under various treatments gradually increased with the growth stage (Fig. 5B). At each stage, with the increasing Cleome spinosa fumigation dose, the SOD activity in cucumber leaves first gradually increased, and when the fumigation dose reached 6.0 g kg⁻¹, the SOD activity no longer significantly increased. The SOD activity at Z2, Z3, Z4 and Z5 treatments were significantly higher than that of the control at each stage. The SOD activity in cucumber leaves increased by 139.5%, 123.9%, and 120.9% at seedling, flowering, and fruit stages, respectively, compared to the control, under the Z4 treatments. Under the control treatment, the SOD activity in cucumber leaves at seedling, flowering, and fruit stages was 51.6 U g⁻¹ min⁻¹, 59.1 U g⁻¹ min⁻¹ and 64.5 U g⁻¹ min⁻¹, respectively; whereas the SOD activity at three stages was 123.6 U g⁻¹ min⁻¹, 132.3 U g⁻¹ min⁻¹ and 142.5 U g⁻¹ min⁻¹, respectively, under the Z4 treatment.

At each stage, with an increase in the fumigation dose of Cleome spinosa, the POD activity in cucumber leaves first gradually increased, and when the fumigation dose reached 6.0 g kg⁻¹, the SOD activity no longer increased (Fig. 5C). The POD enzyme activity in cucumber leaves at Z1, Z2, Z3, Z4 and Z5 treatments was significantly higher than that of the control at all stages. The POD enzyme activity in cucumber leaves increased by 226.1%, 258.3%, and 216.1% at seedling, flowering, and fruit stages, respectively, compared to the control at Z4 treatment. Under the control treatment, the POD enzyme activity in cucumber leaves was 46 U g⁻¹ min⁻¹, 48 U g⁻¹ min⁻¹ and 62 U g⁻¹ min⁻¹ at seedling, flowering, and fruit stages, respectively, whereas the POD activity at three stages was 150 U g⁻¹ min⁻¹, 172 U g⁻¹ min⁻¹ and 196 U g⁻¹ min⁻¹, respectively, under the Z4 treatment.

Effects of Cleome spinosa fumigation on glycolysis related enzyme activity of cucumber

Under each treatment, the hexokinase activity of cucumber leaves gradually increased over time, while at each stage, the hexokinase activity of cucumber leaves continued to increase with the increase in the fumigation dose of Cleome spinosa (Fig. 6A). When the fumigation dose reached 6.0 g kg⁻¹, the hexokinase activity of cucumber leaves no longer significantly increased. The activity of hexokinase in cucumber leaves at Z1, Z2, Z3, Z4 and Z5 were significantly higher than that in the control at all stages. The activity of hexokinase in cucumber leaves increased by 292.3%, 266.7%, and 176.2% compared to the control at seedling, flowering, and fruit stages, respectively. Under the control treatment, the hexokinase activity of cucumber leaves was 1.3 U g⁻¹ min⁻¹, 1.5 U g⁻¹ min⁻¹ and 2.1 U g⁻¹ min⁻¹ at seedling, flowering and fruit stages, respectively; while the hexokinase activity of cucumber leaves was 5.1 U g⁻¹ min⁻¹, 5.5 U g⁻¹ min⁻¹ and 5.8 U g⁻¹ min⁻¹ at seedling, flowering, and fruit stages, respectively.

Fig. 6.

Effect of different doses of Cleome spinosa fumigation on glycolysis related enzyme activity of cucumber. a Hexokinase activity. b Pyruvate kinase activity. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

Under each treatment, the pyruvate kinase activity of cucumber leaves increased with the growth stage, while the pyruvate kinase activity of cucumber leaves increased with the increase in the fumigation dose of Cleome spinosa at each growth stage (Fig. 6B). When the fumigation dose reached 6.0 g kg⁻¹, the pyruvate kinase activity no longer significantly increased. The activity of pyruvate kinase in cucumber leaves at Z1, Z2, Z3, Z4 and Z5 were significantly higher than that in the control at all stages. Under the Z4 treatment, the pyruvate kinase activity of cucumber leaves was 171.4%, 150.0%, and 131.8% higher than that of the control at seedling, flowering, and fruit stages, respectively. Under the control treatment, the pyruvate kinase activity of cucumber leaves was 0.14 U g⁻¹ min⁻¹, 0.18 U g⁻¹ min⁻¹ and 0.22 U g⁻¹ min⁻¹ at seedling, flowering, and fruit stages, respectively; the pyruvate kinase activity of cucumber leaves under the Z4 treatment at seedling, flowering, and fruit stages was 0.38 U g⁻¹ min⁻¹, 0.45 U g⁻¹ min⁻¹ and 0.51 U g⁻¹ min⁻¹, respectively.

Effects of Cleome spinosa fumigation on tricarboxylic acid cyclase activity of cucumber

Under each treatment, the succinate dehydrogenase activity of cucumber leaves increased with the growth process; while at each growth stage, the succinate dehydrogenase activity of cucumber leaves gradually increased with the increase of fumigation dose of Cleome spinosa (Fig. 7A). Succinate dehydrogenase activity in cucumber leaves at Z1, Z2, Z3, Z4 and Z5 treatments were significantly higher than that in the control at all stages. The succinate dehydrogenase activity of cucumber leaves under the Z5 treatment increased by 201.2%, 134.8%, and 112.4% compared to the control during the seedling, flowering, and fruit stages, respectively. Under the control treatment, the succinate dehydrogenase activity of cucumber leaves was 8.4 U g⁻¹ min⁻¹, 11.2 U g⁻¹ min⁻¹ and 13.7 U g⁻¹ min⁻¹ during the seedling, flowering, and fruit stages, respectively. Under the Z5 treatment, the succinate dehydrogenase activity of cucumber leaves was 25.3 U g⁻¹ min⁻¹, 26.3 U g⁻¹ min⁻¹ and 29.1 U g⁻¹ min⁻¹ during the seedling, flowering, and fruit stages, respectively.

Fig. 7.

Effect of different doses of Cleome spinosa fumigation on tricarboxylic acid cyclase activity of cucumber. a Succinate dehydrogenase. b Malic dehydrogenase. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

Under each treatment, the activity of malic dehydrogenase in cucumber leaves increased with the growth process; while the activity of malic dehydrogenase in cucumber leaves at each growth stage continued to increase with the increase of fumigation dose of Cleome spinosa (Fig. 7B). The activity of malic dehydrogenase in cucumber leaves at Z1, Z2, Z3, Z4 and Z5 treatments were significantly higher than that of the control at all stages. The activity of malic dehydrogenase in cucumber leaves under the Z5 treatment increased by 398.2%, 345.3%, and 275.9% compared to the control during the seedling, flowering, and fruit stages, respectively. Under the control treatment, the activity of malic dehydrogenase in cucumber leaves was 5.5 U g⁻¹ min⁻¹, 6.4 U g⁻¹ min⁻¹ and 8.3 U g⁻¹ min⁻¹ during the seedling, flowering, and fruit stages, respectively. Under the Z5 treatment, the activity of malic dehydrogenase in cucumber leaves during seedling, flowering, and fruit stages was 27.4 U g⁻¹ min⁻¹, 28.5 U g⁻¹ min⁻¹ and 31.2 U g⁻¹ min⁻¹, respectively.

Effects of Cleome spinosa fumigation on ATPase activity of cucumber

Under various treatments, the Mg²⁺-ATPase activity of cucumber leaves gradually increased with the growth process of the cucumber, while at each growth stage, the Mg²⁺-ATPase activity of cucumber leaves increased with the increase of fumigation dose of Cleome spinosa (Fig. 8A). The Mg²⁺-ATPase activity of cucumber leaves at Z3, Z4 and Z5 treatments was significantly higher than that of the control at all stages. Under the Z5 treatment, the Mg²⁺-ATPase activity of cucumber leaves was significantly higher than that of the control at all stages. Under the control treatment, the Mg²⁺-ATPase activity of cucumber leaves was 0.38 U g⁻¹ min⁻¹,0.43 U g⁻¹ min⁻¹ and 0.48 U g⁻¹ min⁻¹ during the seedling, flowering, and fruit stages, respectively. Under the Z5 treatment, the activity of Mg²⁺-ATPase in cucumber leaves was 0.70 U g⁻¹ min⁻¹, 0.74 U g⁻¹ min⁻¹ and 0.85 U g⁻¹ min⁻¹ during the seedling, flowering, and fruit stages, respectively.

Fig. 8.

Effect of different doses of Cleome spinosa fumigation on ATPase activity of cucumber. a Mg²⁺-ATPase activity. b Ca²⁺-ATPase activity. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

Under various treatments, the Ca²⁺-ATPase activity of cucumber leaves gradually increased with the growth process of the cucumber, while at each growth stage, the Ca²⁺-ATPase activity of cucumber leaves gradually increased with the fumigation dose of Cleome spinosa (Fig. 8B). After the fumigation dose reached 6.0 g kg⁻¹, the Ca²⁺-ATPase activity of cucumber leaves no longer significantly increased. Both Z4 and Z5 significantly increased the Ca²⁺-ATPase activity of cucumber leaves. The Ca²⁺-ATPase activity of cucumber leaves increased by 62.1%, 23.8% and 21.3% compared to the control during the seedling, flowering, and fruit stages, respectively. Under the control treatment, the Ca²⁺-ATPase activity of cucumber leaves was 0.29 U g⁻¹ min⁻¹, 0.42 U g⁻¹ min⁻¹ and 0.47 U g⁻¹ min⁻¹ during the seedling, flowering, and fruit stages, respectively. Under the Z5 treatment, the Ca²⁺-ATPase activity of cucumber leaves was 0.47 U g⁻¹ min⁻¹, 0.52 U g⁻¹ min⁻¹ and 0.57 U g⁻¹ min⁻¹ during the seedling, flowering, and fruit stages, respectively.

Effects of Cleome spinosa fumigation on number of rhizosphere microorganisms of cucumber

Among all treatments, the Z0 treatment had the highest number of fungi, with 9.56 × 10⁵ per gram of soil, significantly 82.1% higher than the CK treatment; The CK and Z0 treatments have the highest number of bacteria, with 4.82 × 10⁶ and 4.86 × 10⁶ per gram of soil, respectively. The L treatment significantly reduced the number of bacteria and fungi in cucumber rhizosphere soil. Under this treatment, the number of bacteria and fungi per gram of soil was 1.48 × 10⁶ and 1.52 × 10⁵, respectively. Under the L treatment, the bacterial count in cucumber rhizosphere soil decreased by 69.3% and 69.5% respectively compared to the CK and Z0 treatments, and the fungal count decreased by 71.0% and 84.1% respectively compared with the CK and Z0 treatments. The fumigation treatment of different concentrations of Cleome spinosa fragments significantly reduced the bacterial count in the cucumber rhizosphere soil. Moreover, the bacterial count gradually increased with the fumigation dose of Cleome spinosa fragments. When the fumigation dose of Cleome spinosa fragments reached 6.0 g kg⁻¹, the bacterial count no longer significantly decreased. Under the Z4 treatment, the bacterial count in the rhizosphere soil of cucumber was 1.45 × 10⁶ per gram, decreased by 69.9% and 70.2% compared to the CK and Z0 treatments, respectively. The fungal count in the cucumber rhizosphere soil under fumigation with different concentrations of Cleome spinosa fragments was significantly lower than that of the control. Among them, the fungal count under the Z1 treatment was higher than that of the CK, while the fungal count under Z2, Z3, Z4 and Z5 treatments was significantly lower than that of CK. As the fumigation dose of Cleome spinosa fragments increased, the number of fungi in the cucumber rhizosphere soil showed a similar downward trend as the number of bacteria. After the fumigation dose of Cleome spinosa fragments reached 6.0 g kg⁻¹, the number of fungi no longer significantly decreased. Under the Z4 treatment, the number of fungi in the rhizosphere soil of cucumber is 1.47 × 10⁵ per gram, decreased by 72.0% and 84.6% compared to the CK and Z0 treatments, respectively (Fig. 9).

Fig. 9.

Effect of different doses of Cleome spinosa fumigation on number of rhizosphere microorganisms of cucumber. a Bacterial number. b Fungal quantity. Each histogram stands for mean ± SD from 3 duplicates. Those diverse little letters stand for difference of statistical significance (P < 0.05)

Discussion

Angus et al. (1994) crushed and uniformly mixed Brassica plants in the soil of wheat fields, significantly reducing the number of wheat take-all pathogens (Gaeumannomycos graminis) in the soil. For the first time, this measure that can effectively control soil borne diseases was named biological fumigation. In recent years, many scholars have studied and reported on the inhibitory effect of plants on plant pathogens. Using biological fumigation to control soil harmful organisms can effectively reduce the application of chemically synthesized pesticides, as well as reduce pesticide residues and drug resistance, thereby reducing environmental pollution and achieving the goal of green prevention and control of plant soil borne diseases. Biological fumigation has a good control effect on some soil borne plant pathogens, which can replace some chemical pesticides to reduce their usage, increase crop yield, and better promote sustainable agricultural development. Brassica plants can be used as green manure in the field, which can reduce the incidence of Pythium by 20% (Mattner et al. 2008). Wang et al. (2009) reported that Brassica plants can significantly reduce the number of Verticillium wilt and root knot nematodes in the field. In this study, different doses of Cleome spinosa significantly reduced the disease index of cucumber Fusarium wilt, indicating that fumigation of drunk Cleome spinosa has a good alleviating effect on cucumber Fusarium wilt disease.

Plant height can reflect the growth rate of the plant, while stem thickness can reflect the robustness of the plant. The formation of crop yield mainly depends on the accumulation and distribution of dry matter, with dry matter as the material basis (Zou et al. 2021). In this study, the fumigation treatment of 7.5 g kg⁻¹ Cleome spinosa significantly increased the plant height and dry weight of cucumber plants, indicating that Cleome spinosa themselves have a promoting effect on cucumber growth.

Photosynthetic pigments are important substances in plant photosynthesis, and the amount of chlorophyll content can affect the photosynthetic capacity of plants (Zou et al. 2018). In this study, fumigation with 7.5 g kg⁻¹ Cleome spinosa significantly increased the leaf chlorophyll content of cucumber plants, indicating that Cleome spinosa can promote photosynthetic pigments synthesis in cucumber. In addition, in this study, the fumigation treatment of 7.5 g kg⁻¹ Cleome spinosa significantly increased the Vc, free amino acid, soluble protein content, and individual plant yield of cucumber fruits, indicating the role of Cleome spinosa in improving cucumber yield and fruit quality.

Plants maintain a relatively high level of antioxidative activity under environmental stress to eliminate ROS and reduce damage (Parihar et al. 2015). In order to avoid oxidative damage by ROS, antioxidants, including key enzymes (i.e., SOD, POD, CAT, APX, and GR) catalyze ROS detoxification (Vuleta et al. 2016). In this study, cucumber leaves were significantly increased in CAT, SOD and POD activities after fumigation with different doses of Cleome spinosa. These results indicate that after fumigation treatment with Cleome spinosa, the protective enzyme system of cucumbers quickly responds to maintain a balance of intracellular reactive oxygen species metabolism, keeping oxygen free radicals at a lower level, avoiding damage to biofilms by reactive oxygen species, and enhancing plant disease resistance.

The glycolytic pathway is an important process in respiratory metabolism and glycolysis. In adverse environments, plants need to enhance their resistance by consuming a large amount of energy. Glycolysis pathway is an important way of energy generation. In the glycolysis pathway, hexokinase, phosphofructose kinase and pyruvate kinase are the most important irreversible enzymes (O’Leary and Plaxton 2020). Glucose is catalysed by hexokinase to 6-phosphate glucose, hexose isomerase acts on 6-phosphate glucose and converts it into 6-phosphate fructose, then reacts with phosphofructose kinase to 1-6-diphosphate fructose, and then converts phosphoenolpyruvate into pyruvate and ATP under the action of pyruvate kinase (Igamberdiev and Kleczkowski 2018). In this study, the activities of hexokinase and pyruvate kinase in cucumber leaves were significantly increased under the fumigation treatment of different doses of Cleome spinosa, and the enzyme activity increased with the increase of fumigation dosage. This indicated that fumigation of p Cleome spinosa can effectively regulate the glycolysis of cucumber, thereby promoting the decomposition of sugars and the accumulation of pyruvate, so as to enhance the resistance of cucumber to biotic stress such as cucumber Fusarium wilt. The tricarboxylic acid cycle is an equally important sugar metabolism process as glycolysis. In the tricarboxylic acid cycle, malate dehydrogenase and succinate dehydrogenase are key enzymes (Medeiros et al. 2021). In this study, the activities of succinate dehydrogenase and malic dehydrogenase in cucumber leaves were significantly increased under fumigation treatments of 1.5, 3.0, 4.5, 6.0, and 7.5 g kg⁻¹ Cleome spinosa. These results indicated that fumigation of drunk butterfly pollen fragments can promote the tricarboxylic acid cycle in cucumbers, providing energy for cucumbers to resist bacterial invasion.

The Ca²⁺-ATPase and Mg²⁺-ATPase on the cell membrane play an important role in maintaining physiological metabolic balance in both the body and external environment of life. Ca²⁺-ATPase can fine tune intracellular Ca²⁺ balance through active transport (Yadav 2021). In this study, fumigation treatment with 6.0 and 7.5 g kg⁻¹ Cleome spinosa significantly increased the activities of Mg²⁺-ATPase and Ca²⁺-ATPase in cucumber leaves, implying that Cleome spinosa fumigation can maintain physiological and metabolic balance inside and outside cucumber cells by regulating the activities of Mg²⁺-ATPase and Ca²⁺-ATPase, thereby enhancing its resistance to cucumber wilt disease.

Author contributions

XZ and DL performed the overall experiment and data analysis. XM, XJ, RS, ZW, JL, BW and CZ performed the experiment to confirm the results and wrote the manuscript together. YZ designed and managed whole experiments and finalized the manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interests.