Study on the use of Imazalil to continuous cropping obstacle of Ganoderma lucidum caused by Xylogone ganodermophthora

Background The Xylogone ganodermophthora is a pathogenic bacterium that poses a significant challenge to the continuous cultivation of the Ganoderma lucidum fungus . This study aims to identify and investigate specific agents for the effective prevention and control of X. ganodermophthora , establishing a theoretical foundation for over-coming this persistent challenge in G. lucidum cultivation. Results Using different G. lucidum soil as materials to study the presence of X. ganodermophthora in the soil. Additionally, the plate confrontation test was employed to investigate the impact of X. ganodermophthora on G. lucidum growth. The impact of physical factors and antibacterial agents on pathogenic bacteria was successfully carried out, with a further exploration of the effectiveness of field control. PCR amplification experiment and sequencing analysis verified that X. ganodermophthora existed in G. lucidum continuous cropping obstacle soil. This pathogenic bacteria has a significant inhibitory effect on the growth of G. lucidum , with an inhibition rate of up to 52.23%. High temperature, low temperature, light and other physical factors have no obvious inhibitory effect on this pathogen. Further investigation revealed that specific drugs, such as low concentrations (10 μL/mL) of Acticide DB20 and Imazalil, could effectively inhibit X. ganodermophthora growth in G. lucidum. Among them, Imazalil has a notable inhibitory effect on the growth of X. ganodermophthora. Conclusions Indoor toxicity test and field control results showed that Imazalil could effectively control the growth of pathogen X. ganodermophthora in G. lucidum continuous cropping obstacle, and promote the growth of G. lucidum.


Introduction
Ganoderma lucidum, also known as fairy grass, belongs to Ganoderma of Basidiomycetes, Polyporaceae.It is a kind of large fungi with both medicinal and edible functions [1].Historical texts such as Shen Nong Ben Cao Jing, Re-repair of politics and history of the standby Materia Medica, and Ben Cao Gang Mu have documented the positive effects of G. lucidum on mental energy, blood production, mind relaxation, and the treatment of deafness.G. lucidum has been extensively utilized in China, Japan, Korea, the Philippines, Malaysia, Singapore, and Indonesia [2,3].Following the inclusion of G. lucidum fruiting bodies as legal Chinese medicinal materials in Chinese Pharmacopoeia [2,3], G. lucidum was included in American Herbal Pharmacopoeia and Therapeutic Compendium in 2010 [4].However, there is a limited supply of wild G. lucidum, meaning that the natural supply falls short of the increasing market demand.Therefore, the artificial cultivation of G. lucidum industry has also emerged.Artificial cultivation has caused the production of G. lucidum to increase year by year.Based on data, from 2010 to 2020, there was a significant increase in the production of G. lucidum in China, with the output increasing from 91,200 tons to 190,000 tons.Industrial cultivation of G. lucidum has contributed significantly to the growth of the industry.Currently, there are various G. lucidum processing products available in the market, including G. lucidum spore powder and granules [5].
In the process of planting G. lucidum in farmland, a common issue that arises after 2-3 years is a continuous cropping obstacle, "Ganoderma lucidum continuous cropping obstacle" refers to the phenomenon that G. lucidum is continuously planted in the same soil area during the artificial cultivation process, and even under normal cultivation and management conditions, the growth becomes weak, the pests and diseases intensify, and the yield decreases [6].This phenomenon is influenced by various factors, such as shifts in soil biochemical factors, changes in soil microbial population characteristics, autotoxicity of crop root products, and the proliferation of certain pathogenic bacteria [7,8].Currently, soil disinfection and sterilization methods can somewhat alleviate the continuous cropping obstacle for G. lucidum.However, it can also lead to soil hardening, fertility decline, and weakened resistance [9].Therefore, there is an urgent need to identify a viable solution for the ongoing production of G. lucidum to allow for continued growth of the G. lucidum industry.
Liu et al. [10] isolated and purified a pure culture of ascomycete when studying continuous cropping obstacle of G. lucidum grown on basswood, and determined it as X. ganodermophthora by morphological observation and molecular identification.Since Kang et al. [11] first discovered this fungus on G. lucidum yellow rot and confirmed its pathogenicity to G. lucidum [11], Tong et al. [12] found this fungus in associated fungi of G. lucidum in Cambodia [12].Typical symptoms include the internal tissues at the base of the G. lucidum or the tissues within the basswood that have grown G. lucidum and been infected turn yellow.No or only a few fruiting bodies formed, or the pileus growing on the infected section of wood was malformed.Yellow rot can cause yield reduction, limit the continuous cultivation in the same location, and it requires the cultivation site to be changed on the third year following two years of inoculation.The above results indicate that there is a concomitant relationship between X. ganodermophthora and the growth of G. lucidum, and it may be one of the pathogens of Ganoderma lucidum continuous cropping obstacle, and may transmit pathogenicity through cultivated soil.
The purpose of this experiment is to verify whether X. ganodermophthora exists in G. lucidum continuous cropping soil, explore its influence on the growth of ten G. lucidum varieties, and at the same time to prevent it, to find a new way to solve G. lucidum continuous cropping obstacles.

Soil samples and conservation
Samples were taken at four locations on January 13, 2021.CK was taken from random soil near the first dining hall of Fujian Agriculture and Forestry University (26.093534°N, 119.242990°E), GL0 was taken from the wild soil near the Organic Ganoderma Expo Park in Nanping City, Pucheng County, Fujian Province, GL2 and GL4 (27.928986°N, 118.526909°E) were taken from the soil planted with Ganoderma lucidum for 2 years and G. lucidum for 4 years in the Organic Ganoderma Expo Park.The straight-line distance between CK and GL2 is about 221.2 km, GL2 and GL4 are located in different greenhouses in the same park, the straight-line distance is about 113 m, and the straight-line distance between GL4 and GL0 is about 1.4 km.Four experimental soils, each with 3 biological replicates, each sample about 100 g, were stored at − 20℃.

Soil DNA extraction and detection
G. lucidum soil DNA was extracted from different groups, including the CK group, GL0 group, GL2 group, and GL4 group, following the instructions provided by the soil DNA extraction kit (OMEGA).There were four groups, each group repeated three times.The concentration and purity were determined using a nucleic acid microanalyzer.
The inhibitory effect of X. ganodermophthora on the growth of G. lucidum mycelium Set X. ganodermophthora confrontation group and G. lucidum control group.G. lucidum control group was inoculated G. lucidum only on the PDA plate, in the confrontation group, X. ganodermophthora was inoculated 3 cm away from G. lucidum.Next, colony diameter was measured and the suppressive impact of X. ganodermophthora was measured to determine the inhibition rate on G. lucidum mycelium growth.

Indoor toxicity measurement
Indoor toxicity was determined using the growth rate method.Three types of concentration gradients are set for each agent in The fungus packs were then positioned within a greenhouse covered by a shading net that provided 90% shade.Proper ventilation was ensured, the carbon dioxide concentration was maintained at the same level as the surrounding atmosphere, and air humidity was maintained at approximately 70%.Upon opening each fungus pack, we ensured that it was covered with a layer of exposed soil (2-3 cm in thickness).The blank control group, low-dose control group, and high-dose control group were treated with 20 mL of water every Wednesday.Conversely, the tie-back test group, low-dose experimental group, and high-dose experimental group were given 20 mL of pathogenic bacterial liquid.Additionally, the blank control group and tie-back test group received 10 mL of water every Sunday.Low-dose control group and low-dose experimental group received low-dose medicine 10 mL every Sunday.Similarly, the medium-dose control group and medium-dose experimental group received 10 mL of

Statistical analyses
Data were subjected to one-way analysis of variance (ANOVA), and the mean values indicating statistical significance were compared by Duncan's multiplerange test using SPSS 25.These data are all expressed as the mean ± standard deviation.P < 0.05 were considered to be statistically significant.

Detection of X. ganodermophthora in soil utilized for continuous cultivation of G. lucidum
Previous studies [10][11][12] have provided evidence that X. ganodermophthora is a pathogenic fungus that infects and hinders the growth of G. lucidum.
As shown in Fig. 1. Results showed that soil DNA of CK and GL0 groups could not amplify bands, but soil DNA of GL2 and GL4 groups could amplify bands.
After sequencing and Genbank alignment analysis, the band sequence was highly homologous to X. ganodermophthora in Xylogone, indicating that X. ganodermophthora existed in continuous cropping soil, and X.
ganodermophthora was related to continuous cropping obstacle of G. lucidum.

Experimental investigation into the impact of pathogenic bacteria on the growth inhibition of G. lucidum during continuous cultivation
Figure 2 shows that on the third day of the confronting culture, the hyphae of G. lucidum and G. sinense started to make contact with the hyphae of X. ganodermophthora.By the fifth day, the hyphae of ten species of G. lucidum had been in contact with the X. ganodermophthora hyphae (Table 2).By calculating the inhibition rate of pathogenic fungus hyphae on G. lucidum hyphae in 5 days, it was found that X. ganodermophthora grows extremely fast.It has significant inhibition effect on mycelium growth of 10 different G. lucidum strains, more than half of the tested G. lucidum strains showed an inhibition rate over 40%.Among them, the inhibition rate of Ganoderma leucocontextum 1 is 52.63%.Taken together, these data clearly showed that X. ganodermophthora inhibits the growth of G. lucidum strains.

Impact of physical conditions on X. ganodermophthora proliferation
As shown in Fig. 3, in terms of growth temperature, the growth temperature of G. lucidum mycelium ranges from 4 to 35 °C, with an optimal temperature of 24-28 °C.Beyond this temperature range, the mycelium stops growing or exhibits abnormal growth or even death.However, pathogenic bacteria can maintain rapid growth at 25-32 °C, with an optimal growth temperature of 28 °C, which is the same temperature for optimal growth of G. lucidum mycelium.Regarding pH, different mycelium varieties grown on PDA plate culture medium display significant differences, what they have in common is that they can grow in the range   of 4-10 pH, and the optimum growth pH is 5-7 [13].However, we found that the mycelia of pathogenic bacteria maintained rapid growth between pH 2-9, and that growth was fastest at pH 5.In terms of light, the fruit body of G. lucidum is sensitive to light and exhibits light-oriented growth [14].Our data showed that the pathogen was also conducive to mycelial growth under 12 h of light.The above results suggest that it is not feasible to physically eliminate X. ganodermophthora according to biological characteristics.

Evaluation of specific inhibitors of pathogenic microorganisms in continuous cropping G. lucidum
The outcomes are presented in Fig. 4. All nine medications were discovered to have inhibitory effects on the growth of X. ganodermophthora.Notably, Acticide DB20 and Imazalil were found to strongly impede the development of X. ganodermophthora even at low concentrations.The suppressive impact of Imazalil was the most notable.The growth of G. lucidum was enhanced by a high concentration of Hachemical CPH ether, as well as low concentrations of Triforine, Vinclozolin, and Imazalil (Fig. 4A and B).This indicates that Triforine, Vinclozolin, and Imazalil have the potential to be used as agents to mitigate the challenges associated with continuous planting of G. lucidum.However, further research and verification are required to confirm these findings.
Following the plate experiment (Fig. 5) we found that the colonies of pathogenic bacteria in the CK group had a white color during the initial phase of growth.Three days later, the hyphae surrounding the inoculation block initiated the release of yellow pigments, and the conidia started to develop.After 6 days, the mycelium fully covered the plate.Nevertheless, when exposed to a low concentration of Imazalil the mycelium of the pathogen exhibited no growth for a duration of 7 days.Compared with the control group, the G. lucidum plate mycelium treated with a low concentration of Imazalil showed an improved growth rate and density.The plate experiment demonstrated that a low dose of Imazalil effectively suppressed the growth of the pathogen's mycelium, while simultaneously stimulating G. lucidum mycelium growth.

Effect of Imazalil on fruiting bodies of G. lucidum
As shown in Fig. 6 and Table 3.The growth cycle of G. lucidum in the first tide is 35 days.The growth rate of G. lucidum in the tie-back test group showed a noticeable decrease, resulting in a prolonged growth cycle.The G. lucidum count in a single pack of the tie-back test group, low-dose experimental group, and medium-dose experimental group also increased while the width decreased.The low-dose control group showed a 0.44% increase in bag output compared to the blank control group, whereas the medium-dose control group showed a 1.14% increase compared to the blank control group.The tie-back test group exhibited a 6.96% decrease in the production of a single bag compared to the blank control group.Under the action of Imazalil, the low-dose experimental group and the medium-dose experimental group showed an increase of 1.92% and 2.09% in output, respectively, compared to the tie-back test group.The first tide of G. lucidum data demonstrated that Imazalil has a specific stimulating impact on the growth of G. lucidum, aligning with the findings of the plate experiment.
As shown in Fig. 7 and Table 3.The growth cycle of G. lucidum in the second tide is 45 days.The number of single bag of G. lucidum increased significantly in the tie-back test group, the low-dose experimental group, and the medium-dose experimental group.The number of single bag of G. lucidum in the blank control group, the low-dose control group, and the medium-dose control group also increased slightly.Correspondingly, the length and width of the second tide of G. lucidum in each group noticeably decreased.The low-dose control group showed an approximate 3.53% increase in yield per bag compared to the blank control group, while the high-dose control group showed a 24.92% increase.Conversely, the tie-back test group exhibited a 12% decrease in yield per bag compared to the blank control group.Under the influence of Imazalil, the low-dose and medium-dose experimental groups showed a respective increase in yield per bag of 28.80% and 47.65% compared to the reinoculation experimental group.The data from the second tide of G. lucidum demonstrates that Imazalil has a significant inhibitory effect on X. ganodermophthoras growth.

Conclusions
Currently, the issue of continuous cropping obstacles in G. lucidum cultivation represents a primary constraint impeding the development of the G. lucidum industry [15,16].The persistent challenge of continuous farming in G. lucidum is commonly attributed to alterations in soil microbial features and the autotoxicity of G. lucidum products [17][18][19].Ma et al. [20] think in continuous cropping soil microbial population, bacteria have strong inhibition on allelopathy of G. lucidum thallus.Among fungi, Trichoderma exhibited the most pronounced inhibitory impact on the growth of G. lucidum among the fungal microorganisms, with penicillium and Streptospora following suit.Further, Zhang et al. [21] believe that autotoxicity can impact the development of plant somatic cells, the permeability of cell membranes, enzyme activity, and the absorption and utilization of nutrients [21], thus affecting the growth of crops, resulting in continuous cropping obstacles.To break through this limitation, many researchers have explored the generation and prevention mechanisms of G. lucidum continuous cropping disorder to improve the issue of G. lucidum continuous cropping disorder.Ja et al. [22] have employed different bacteriostatic agents to treat G. lucidum and hinder the growth of harmful bacterial hyphae.Wu et al. [23] fumigated continuous cropping soil with liquid ammunition.Yuan et al. [24] treated continuous cropping soil with lime soaking and uniform sprinkling.All of the above can improve the continuous cropping obstacle of G. lucidum.
Imazalil, a compound that inhibits the production of sterols, was granted approval for use in 1979 [25][26][27].The primary mechanism by which it exhibits its antibacterial properties is through the inhibition of ergosterol production.It is mostly employed as a bacteriostatic agent or for fruit preservation [28][29][30].Imazalil has high sensitivity and good control effect on pathogenic bacteria such as wet blister disease and crown rot [31][32][33].There have been many studies on the effects of G. lucidum continuous cropping obstacles on soil microorganisms, one of which is X. ganodermophthora.Hence, this investigation uses Imazalil as a means to inhibit and manage X. ganodermophthora.These findings demonstrated that Imazalil has a substantial inhibitory impact on the X. ganodermophthora growth.
The specific primers used to amplify the soil samples showed that the DNA from the CK group and GL0 group did not produce any amplified bands, however the DNA from the GL2 group and GL4 group successfully produced amplified bands.Simultaneously, the findings of the flat plate confrontation test demonstrated a notable inhibitory impact of X. ganodermophthora, a decomposing fungus of G. lucidum, on the growth of G. lucidum hyphae.Based on the aforementioned investigations, we concluded that X. ganodermophthora, a microorganism that causes spoiling in G. lucidum, is one of the pathogenic bacteria that hinders the ongoing cultivation of G. lucidum.Through the examination of the biological attributes of pathogenic bacteria, our aim was to investigate potential physical methods for preventing and managing these germs, as well as identifying specific inhibitors to effectively achieve the goal of prevention and control.This pathogen's hyphae exhibit vigorous development within a temperature range of 25-32 °C.High temperature, low temperature, total darkness and total light could h not effective for the growth of X. ganodermophthora hyphae.The hyphae become inactive at a temperature of 60 °C or at pH 2. Indoor control agent screening revealed that even at low concentrations, Imazalil can effectively hinder the growth of X. ganodermophthora and stimulate the growth of G. lucidum.Here, we tested the effectiveness of imazalil in controlling continuous crop growth through field experiments.
In the early stage of laboratory, ITS1 amplicon sequencing was carried out on adjacent wild soil, 1-year, 2-year and 4-year cultivated G. lucidum soil by Illumina MiSeq platform.The results revealed a decrease in fungal diversity in the G. lucidum cover soil as the number of consecutive cropping years increased.The covering soil of G. lucidum planted for four years only contained Basidiomycetes, Ascomycetes and a small amount of Mortierella.The relative abundance of fungi in soil changed greatly with the continuous cropping years, among which Basidiomycota increased significantly, Ascomycota decreased significantly, but Mortierella had no significant difference.In the previous experiment, 150 m 2 of soil planted for four years were treated with 45 kg of lime.The disease rate of G. lucidum can be reduced by about 28.28%, significantly improving G. lucidum diseases.At the same time, the lime soaking treatment of Ganoderma lucidum continuous cropping soil changed the fungal structure in the covering soil, which was mainly manifested in the decrease in the relative abundance of Ascomycota and the increase in the relative abundance of Basidiomycota [24,34].This investigation established that the utilization of Imazalil effectively suppressed the proliferation of X. ganodermophthora spoiling in G. lucidum.Therefore, we hypothesize that Imazalil along with lime may be a promising strategy to overcome continuous cropping obstacles in G. lucidum and to promote the development of the G. lucidum industry.
(i) Effect of temperature on growth and development of X. ganodermophthora hyphae: 8 mm diameter pathogen block was attached to PDA culture dish with hyphal side down, and the culture temperature gradient was set at 20, 25, 28, 32, 35, 40, 45, 50, 55, 60, 65, 70 ℃.(ii) Effect of pH on growth and development of X. ganodermophthora hyphae: pH of the medium was adjusted to 8 different grades (2, 3, 4, 5, 6, 7, 8, 9, 10, 11) with 0.1 mol/L hydrochloric acid (HCl) and 0.1 mol/L sodium hydroxide (NaOH) solution before sterilization.Inoculate an 8 mm diameter pathogen block into the center of the dish.Subsequently, the dishes were maintained at 25 °C in darkness for 5 days.(iii) Effects of illumination on growth and development of X. ganodermophthor hyphae: The lighting parameters of the constant-temperature incubator were adjusted to three settings: full light, full darkness, and a 12-h cycle of alternating light and dark periods.PDA dishes inoculated with pathogenic bacteria (8 mm in diameter) were placed in incubators with three light modes and incubated at 20 °C for 7 days.Each of the aforementioned treatments were replicated in triplicate.

Fig. 2
Fig. 2 Mycelia's growth inhibition of pathogenic on G. lucidum by disk diffusion, A, C treatment group (The fungus block on the left is G. lucidum strain, and the fungus block on the right is X. ganodermophthora), B, D: control group; strains of figure A and B, from left to right, are G. multipileum, G. tsugae, G. lucidum, G. sinense and G. leucocontextum 2, respectively; strains of figure C and D, from left to right, are G. leucocontextum 1, G. resinaceum, sporeless cultivar of G. lingzh, G. sinense Y2019 and G. leucocontextum 1905, respectively

Fig. 3 Fig. 4
Fig. 3 Biological characteristics of X. ganodermophthora.A Mycelial of growth rate of X. ganodermophthora in different temperature.B Mycelial of growth rate of X. ganodermophthora in different lethal temperature.C Mycelial of growth rate of X. ganodermophthora in different pH.D Mycelial of growth rate of X. ganodermophthora in different illumination duration

Fig. 5
Fig. 5 Effect of imazali on X. ganodermophthora and G. lucidum mycelium growth.A The growth of X. ganodermophthora in group CK.B Effects of low concentration of Imazalil on the growth of X. ganodermophthora.C Growth of G. lucidum in group CK.D The effect of low concentration of Imazalil on the growth of G. lucidum

Fig. 6 Fig. 7
Fig. 6 The growth situation of first tide.A Blank control group; B Low dose control group; C medium dose control group; D tie-back test group; E low dose experimental group; F medium dose experimental group; the picture below is the same

Table 1
. The pathogens were inoculated into PDA medium and a control group was set up.Record the growth rate of the colonies in a 25 °C incubator.The Inhibition rate = Diameter of colony in control group − Diameter of colony in confrontation group Diameter of colony in control group − Diameter of inoculated block × 100% inhibition rate of mycelium growth was determined by indoor mycelium growth rate inhibition method.

Table 1
The dosage of 9 fungicides in test reagent

Table 2 Mycelia
's growth inhibition of pathogenic on G.

Table 3
Agronomic traits of first tide and second tide