Introduction

Proper cultivation practices for oilseed rape, beans, and other major crops are crucial for ensuring food and oil security. Especially in recent years, the occurrence of extreme events, such as the outbreak of the Covid-19 epidemic, extreme weather conditions, and pest outbreaks, has become more frequent. These factors, to some extent, have disrupted the international trade of oilseed crops among different countries. Furthermore, a variety of diseases, such as Sclerotinia sclerotiorum (S. sclerotiorum), downy mildew and bacterial wilt, have significantly impacted the yield of oilseed crops. It was established that molybdenum was an essential trace element for plants in 1939. Since then, scientists have paid much attention to molybdate compounds for their anti-viral properties1,2,3. In 2014, Deepak pioneered the utilization of a polyaniline tungsten-molybdenum phosphate system for the separation of toxic metals from water4. Furthermore, the same research team synthesized and characterized stannous molybdate and discussed its antimicrobial properties5. Numerous other molybdate systems have been studied for their antimicrobial properties6,7,8,9,10.

As a rare natural mineral material, Cu3(MoO4)2(OH)2 (CM) has received great attention due to its excellent magnetic, photocatalytic, and electrochemical properties11. CM thin films can be used as photocatalysts for the photoconversion of CO2 into valuable compounds12. Copper molybdate material has a major inhibitory and bactericidal effect against Escherichia coli (E. coli)13,Full size image

To better analyze the anti-fungi properties of the materials, X-ray photoelectron spectroscopy (XPS) was employed to analyze the chemical state of Cu3(MoO4)2(OH)2 nanoparticles. Figure 4a shows the whole spectrum of Cu3(MoO4)2(OH)2. Figure 4b–d show the deconvolution spectra of Cu 2p, Mo 3d and O1s. Two peaks at 933.0 and 953.0 eV are attributed to the binding energy of Cu 2p3/2 and Cu 2p1/2, respectively, in Fig. 4b. These signals match the literature values of the Cu2+ state. Figure 4c shows two peaks in the Mo 3d spectra at 232.2 eV and 235.3 eV, which coincide with the Mo 3d5/2 and Mo 3d3/2. It can be seen in Fig. 4c that the oxidation state of molybdenum in Cu3(MoO4)2(OH)2 is + 6. In Fig. 4d, the XPS spectrum of O1s reveals a single peak at 531.5 eV, representing the presence of metal–oxygen bonds and the absence of other oxygen defects38. In summary, the valence states of CM NFs follow the stoichiometric ratio that contains Mo6+.

Figure 4
figure 4

(a) Full XPS spectrum of Cu3(MoO4)2(OH)2, (b) High-resolution Cu 2p, and (c) Mo 3d XPS spectra of Cu3(MoO4)2(OH)2 and (d) O1s XPS spectra of Cu3(MoO4)2(OH)2; (e) low binding energy and high binding energy of UPS spectrum and (f) schematic energy level diagram of CM NFs.

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In order to investigate the physical properties of CM NFs, ultraviolet photoelectron spectroscopy (UPS) spectra have been used to characterize the valence band maximum (VBM) of CM NFs, as shown in Fig. 4e–f. It can be observed that the onset energy (Eonset) of CM NFs is 11.3 eV, while the cutoff energy (Ecutoff) is 20.07 eV. The energy reference is set at the Fermi energy. The VBM of CM NFs is determined to be − 12.46 eV. Meanwhile, the work function of CM NFs is − 9.66 eV. Combined with the conduction band minimum (CBM) derived from the VBM and bandgap (i.e., neglecting the exciton binding energy), it can be found that the energy level is 2.8 eV19. The results confirm that CM NFs behave semiconductive properties.

Fungal inhibition effect

After CM NFs of different concentrations interacted with S. sclerotiorum for 36 h, the results of fungal inhibition were shown in Fig. 5A–E. The action process of nanosheets on fungi can be divided into three stages. When the concentration equals 100 ppm, the hyphae circle begins to shrink, indicating that 100 ppm is the minimum inhibitory concentration (MIC) of nanosheets against S. sclerotiorum. The fungistatic zone at this time is 0.14 cm. As the concentration of nanosheets increased, the mycelial circle shrunk significantly, indicating that the degree of inhibition of fungal growth was positively correlated with the concentration of nanosheets. In the concentration range of 100 ppm to 200 ppm, the rate of change of the hyphae circle was the fastest. As the concentration of nanosheets continued to increase, the change rate of the hyphae circle slowed down; especially in the two concentration intervals of 200–250 ppm and 400–500 ppm, the hyphae circle hardly changed. When the concentration reached 1000 ppm, the hyphae completely disappeared, symbolizing that the fungus had lost the ability to proliferate. This concentration can be considered the minimum fungi concentration (MFC) of nanoflakes.

Figure 5
figure 5

(A) The changes of hypha length after Cu3(MoO4)2(OH)2 nanoflakes interacted with S. sclerotiorum for 36 h at different concentrations. The control group was 0 ppm. (B) The relationship between the concentration of Cu3(MoO4)2(OH)2 and the inhibition of fungal hyphae; (C) Outdoor experimental plants sprayed with 0 ppm nanoflakes. (The brown circular spots are fungal spots); plants with nanoparticles sprayed at 40 ppm at the initial flowering stage, respectively; (D) Relationship between spraying concentration and frequency, and incidence of S. sclerotiorum(s. s.); (E) Correlation between spraying concentration and frequency, and incidence of S. sclerotiorum. Data are presented as mean ± standard deviation (s.d.) with the error bars representing the standard deviation.

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Among various inorganic nanoparticles, Cu-based nanoparticles are considered to have antifungal effects39,40,41. The role of nano-metal particles in anti-S. sclerotiorum is to inhibit the development of S. sclerotiorum spores and conidia, eventually leading to the death of fungal hyphae42. It is generally believed that molybdenum-containing compounds exhibit better antifungal properties. The pour plate test results have confirmed this point: the active ingredient in the antifungal test is CM NFs. As shown in Fig. 5B, four parameters are the maximum hyphal length a, the minimum hyphal length b, the average length of the hypha (a-b)/2 and the anti-fungi zone 1.36-(a-b)/2, respectively. The growth potential range of S. sclerotiorum refers to the growth length of the hypha. The smaller the length of the hypha, the better the inhibition effect on S. sclerotiorum by CM NFs. The anti-fungi area changed drastically in the concentration interval of 100–200 ppm. However, the mycelial growth radius changed little in the concentration ranges of 200–250 ppm and 400–500 ppm, which indicated that the strain had a certain tolerance to higher concentrations of CM NFs.

On the CM NFs—containing cultures, the growth of S. sclerotiorum mycelium was significantly inhibited. As the concentration of nanoflakes increased from 100 to 1000 ppm, the inhibitory effect on fungal growth was gradually enhanced. Similar phenomena have also been observed in other antifungal tests of nano-metal salts23,36,43,44, because metal ions can cause severe damage, including the separation of the cell membranes from the cytoplasm, resulting in cell lysis and impossible germination of sclerotia. Therefore, the antifungal properties of nanoflakes are likely to be related to the semiconducting properties of molybdates inducing the generation of ROS45, which will trigger oxidative stress in cells.

Field planting test

Oilseed rape with a low yield worldwide is mainly due to stem rot resulting from S. sclerotiorum46. In order to further investigate how CM NFs inhibit S. sclerotiorum infection, CM NFs have been sprayed on the oilseed at different growth stages. Zhongyouza 19 was selected as the test sample, and the experiments were carried out in two different experimental sites, referred to as Site 1 and Site 2. At each site, three different concentrations of CM NFs were applied, and each concentration was repeated three times. Therefore, there are nine plots in the experiments, with each plot having an area of 20 m2. In general, the concentration of S. sclerotiorum outside is lower than that in the plate test. The concentrations of CM nanosolutions are 0 ppm (clear water), 40 ppm and 80 ppm, respectively. Then CM nanosolutions were sprayed onto the oilseed rape plants starting from the initial flowering stage. The information is shown in Table 1.

Table 1 The relationship between the spraying stage and spraying concentration.
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After treatment with CM NFs at 0 ppm and 80 ppm at the initial flowering stage (Fig. 5C), the inoculated leaves outdoors show a much weaker influence from s. s. at 80 ppm than the control group treated with water. It is observed that oilseed outside treated with 80 ppm CM NFs grew better and had less infection. The control plants sprayed only with water show severe leaf yellowing and fewer buds, indicating spreading infection of S. sclerotiorum. These findings demonstrated to show that CM NFs at 80 ppm validly inhibit s. s. infection in oilseed rape plants. In the investigation of rape Sclerotiniose, disease severity was calculated statistically based on disease incidence and disease index following the agricultural trade standard (China 2011)47,48. CM NFs were diluted to the specified concentrations with water. Then the diluted solution was sprayed onto the foliage of rapeseed at two experimental sites (Site 1 and Site 2), respectively. Figure 5D indicates that after spraying CM Ns-1 (40 ppm) or CM Ns-2 (80 ppm) only once at the initial flowering stage. The disease index of oilseed rape is reduced by 25.2% or 29.5%, respectively, compared to the control group. If spraying CM Ns-1 (40 ppm) or CM Ns-2 (80 ppm) only once at the rape pod stage, the disease index decreases by -6.2% or 7.8%, respectively. And the disease index has worsened, compared with that at the initial flowering stage. When CM Ns-1 (40 ppm) or CM Ns-2 (80 ppm) is sprayed once at the rape pod stage, and once at the initial flowering stage, the disease index is reduced by 27.8% or 37.3% on average.

The results of the disease index at test Site 2 show that it is significantly reduced by 20.9% or 38.8% with spraying CM Ns-1 (40 ppm) or CM Ns-2 (80 ppm) once at the initial flowering stage. The disease index is reduced by 25.0% or 10.1% by spraying CM Ns-1 (40 ppm) or CM Ns-2 (80 ppm) once at the rape pod stage. The disease index is reduced by 6.1% when CM Ns-2 (80 ppm) is sprayed at the initial flowering stage and the rape pod stage. The most considerable reduction of disease index is 40.9% when CM Ns-1 (40 ppm) is sprayed once at the initial flowering stage and once at the rape pod stage.

Based on the results obtained from Site 1 and Site 2, it can be concluded that CM NFs effectively inhibit infection, leading to a decrease in the disease index of oilseed rape overall. Spraying CM Ns-2 (80 ppm) once at the initial flowering stage, the disease index reduces by 34.2% on average. Spraying CM Ns-1 (40 ppm) once at the initial flowering stage and once at the rape pod stage, the disease index decreases by 34.3% on average. So, both treatments of CM NFs could effectively reduce the occurrence of S. sclerotiorum disease in rapeseed. The incidence of the disease is reduced. On the contrary, the oilseed rape sprayed with water is more seriously infected by S. sclerotiorum.

In Fig. 5E, it can be observed that the inhibition effect from CM Ns-1(40 ppm, lower concentration) sprayed one time at the initial flowering stage and one time at the rape pod stage, is similar to that at CM Ns-2 (80 ppm, high concentration) sprayed only once at initial flowering stage. Their inhibition rates both exceed 34%. Thus, the effect from CM nano solution at low concentrations shows good inhibition on S. sclerotiorum.

On the other hand, copper ions may move into the interior of fungal cells or attach to their outer surfaces, leading to apoptosis through protein denaturation and cell membrane disruption and have more active sites to encounter reduction reactions and yield better antioxidant activity49. The mechanism of this tolerance to copper ions has also been found in experiments on other fungi50; that is, a relatively high concentration of copper ions can stimulate the growth of sclerotia hyphae51. The main explanation for this tolerance mechanism is that S. sclerotiorum has genes that detoxify ROS and copper ions52. The most significant category of radical oxidants (superoxide ion & hydroxyl OH) and non-radicals are the reactive oxygen entities (hydrogen peroxide, organic peroxides). Metal oxide ions are oxidizing species and can trigger ROS production by coupling with hydrogen peroxide or molecular oxygen, contributing to oxidative stress53.

As the concentration of nanoflakes increased to 1000 ppm, which reached the tolerance limit of S. sclerotiorum, the fungus was eliminated, and the hyphae completely disappeared. Due to the smaller particle size of CM NFs, a larger specific surface area is obtained. This increases the probability of copper ions in contact with fungal cells, thereby enhancing the antifungal effect compared to non-nano copper molybdate. Worth noting that the CM NFs are slightly soluble in water and have little impact on the environment.

In addition, since the control field and the test field are adjacent to each other and CM NFs do not exist in the control field, the test field is subjected to repeated infection by S. sclerotiorum from the control field throughout the growth cycle. Since the inherent instability of CM in maintaining a nano-sized structure over an extended period, the enhanced inhibitory effect may also be related to ROS and other factors. Because CM was sprayed on a cloudy day, it is likely that ROS exists to some extent. On the other hand, it is known that CM contains molybdenum, which is also an essential trace element for plant growth. Therefore, it is worthwhile to investigate further whether CM NFs also play a role in enhancing the immunity of rapeseed.