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Article

Leaf Fermentation Products of Allium sativum L. Can Alleviate Apple Replant Disease (ARD)

by
Chengwen Yin
1,2,†,
Rong Zhang
1,†,
Yiming Zhao
3,†,
Gongshuai Wang
4,
Chengmiao Yin
1,
Yusong Liu
1,* and
Zhiquan Mao
1,*
1
College of Horticultural Science and Engineering, Shandong Agricultural University, Tai’an 271018, China
2
Xintai Science and Technology Innovation Development Promotion Center, Tai’an 271018, China
3
Department of Modern Agriculture, Linyi Vocational University of Science and Technology, Linyi 276000, China
4
College of Agricultural Science and Technology, Shandong Agriculture and Engineering University, Jinan 251100, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(6), 546; https://doi.org/10.3390/horticulturae10060546
Submission received: 9 April 2024 / Revised: 20 May 2024 / Accepted: 22 May 2024 / Published: 23 May 2024
(This article belongs to the Section Protected Culture)
Browse Figures
Versions Notes

Abstract

:
Apple replant disease (ARD) is a serious threat to newly replanted apple seedlings. The alleviation of ARD is of great significance for the healthy development of the apple industry. In this study, we investigated the effects of leaf fermentation products (LFP) of Allium sativum L. on the replanted soil environment and Malus hupehensis Rehd. seedlings. The results showed that LFP increased biomass accumulation, changed root architecture, increased root anti-oxidant enzyme activity, and decreased root MDA content under replanted conditions. In addition, the application of LFP increased soil nutrients and soil enzyme activity and reduced phenolic acid content. Furthermore, the LFP enriched the number of beneficial bacteria and reduced the number of harmful fungi, which positively affected the soil microbial community structure. Overall, our results demonstrated that LFP of A. sativum L. could alleviate the occurrence of ARD and provide new insights for the reuse of the leaves of A. sativum L. and the prevention of ARD.

1. Introduction

Due to limited land resources and the upgrading of orchards, apple replanting generally occurs in major apple-producing areas, and apple replant disease (ARD) has become a major problem hindering the sustainable development of the apple industry [1]. The manifestations of ARD are root damage and growth inhibition of replanted apple seedlings, fruit quality and yield decline, disease, and insect infestation aggravation, and even causing the orchard to cease production [2,3]. The causes of ARD include the imbalance of the soil microbial community, deterioration of soil physical and chemical properties, and allelopathy induced by the excessive accumulation of phenolic acids in soil [4,5,6].
Biological control includes the use of a variety of beneficial organisms or biologically produced active substances to prevent and control diseases [7]. Compared with other methods, biological control is more efficient, safe, and environmentally friendly. Previous studies of biological control have focused on bacteria and fungi [8]. However, the application of antagonistic plants is also an important form of biological control and has been extensively studied. Among them, Brassica, Asteraceae, and Allium have strong antagonistic functions to pathogenic fungi, which can play a role in alleviating ARD [9,10].
Allium sativum L. is one of the most important horticultural crops [11]. Studies have shown that the mixed cropping, intercropping, or crop rotation of A. sativum L. with cucumber, tomato, or eggplant could effectively reduce the occurrence of pests and diseases [12]. The mixed cropping of A. sativum L. with apple also reduced the degree of disease caused by ARD. The root exudates of A. sativum L. could inhibit a variety of harmful bacteria in soil and reduce pests and diseases. The appropriate concentration of stems, leaves, and root exudates of A. sativum L. has shown an inhibitory effect on Phytophthora capsici [13]. Maize straw fermentation has good application value in improving soil physicochemical properties and increasing soil organic matter content [14]. Previous studies have shown that the application of straw fermentation of A. sativum L. to tomato soil could reduce the number of root-knot nematodes, and sulfocompounds may play a major role in this process [13,15].
China is mainly based on traditional agriculture, which produces a large amount of dry leaves of A. sativum L. every year. However, there is no reasonable recycling scheme for a large amount of straw, which causes serious environmental pollution. It was found that straw can reduce the harm of pathogenic fungi and alleviate the replant disease [16]. However, there are some problems, such as poor effect and slow decomposition rate in the direct application of straw to soil. In addition, different straw application amounts have different effects on soil. In this study, to retain the bactericidal properties of the dried leaves of A. sativum L. and make effective use of them, we explored the effect of leaf fermentation products (LFP) on ARD control, which is of great significance to guide the biological control of ARD and the recycling of discarded dried leaves of A. sativum L. Results showed that LFP of A. sativum L. could improve soil microbial structure and soil environment, and enhance the tolerance of Malus hupehensis Rehd. seedlings to replanted soil. Our study revealed the effects of LFP of A. sativum L. on the soil environment of old apple orchards and replanting M. hupehensis Rehd. seedlings, which provided a theoretical basis for the prevention of ARD.

2. Materials and Methods

2.1. Plant Materials and Treatments

The experiment was carried out in the College of Horticultural Science and Engineering, Shandong Agricultural University. The replanted soil was obtained from an old orchard in Manzhuang Town where Malus x domestica Borkh cv. Red Fuji have been planted for 32 years.
The dried leaves of A. sativum L. were crushed to a diameter of 0.5 cm, and water was added to 75% of the maximum water-holding capacity of dried leaves. Then, the dried leaves were sterilized at 121 °C for 40 min and inoculated with fermentation strains. Each 500 g of dried leaves was mixed with 150 mL of Bacillus subtilis (1 × 109 CFU·mL−1), 150 mL of Bacillus licheniformis (1 × 109 CFU·mL−1), 100 mL of Aspergillus niger (1.2 × 109 CFU·mL−1) and 100 mL of Aspergillus oryzae (1.2 × 109 CFU·mL−1). The C:N was adjusted to 25:1 by adding urea with a mass of 0.63% of dried leaves, and the pH was adjusted to 7.5 by adding CaCO3. The LFP was obtained by aerobic fermentation at 38 °C for 20 days.
The pot experiment was carried out at the National Apple Engineering Technology Research Center Base on the South Campus of Shandong Agricultural University from May to October 2021. M. hupehensis seedlings were used as experimental materials grown in soil in which apple trees have been cultivated for 32 years. A total of 4 treatments were examined: untreated control soil (CK1), methyl bromide fumigation (CK2), 3% of the soil mass of LFP (T1), 5% of the soil mass of LFP (T2). There were 40 seedlings in each treatment, and all groups were unified for normal water and fertilizer management. A compound fertilizer (N-P2O5-K2O, N:P:K = 5:3:2) of 100 g per pot was applied on the 60th day after the seedlings were planted.

2.2. Determination of Plant Growth

The height, ground diameter, and fresh and dry weight of seedlings were measured by ruler, vernier caliper, and electronic scale, respectively, in August. The professional version of the WinRHIZO (2007 edition) root analysis system was used to analyze the root images of all samples, and the root length, total volume, total surface area, and root tip number of the seedlings were determined. Each seedling was a biological replicate, and three biological replicates were analyzed per treatment.

2.3. Determination of Anti-Oxidant Enzyme Activities and MDA Content of Roots

The fresh roots were washed and dried to determine the activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and malondialdehyde (MDA) content in roots according to the method described by Wang et al. [17]. Each seedling was a biological replicate, and three biological replicates were analyzed per treatment.

2.4. Determination of Soil Enzyme

After removing the topsoil, the rhizosphere soil was used to measure soil enzyme activities. The rhizosphere soil was taken from between the roots, and 500 g of rhizosphere soil was taken from each seedling and dried in a cool place. Urease activity was measured using the indophenol blue colorimetric method. The neutral phosphatase activity was determined by the disodium phenyl phosphate colorimetric method. The sucrase activity was measured by the 3,5-dinitrosalicylic acid colorimetry method. The catalase activity was measured by potassium permanganate titration. The soil from one seedling was used as a biological replicate, and three biological replicates were analyzed for each treatment.

2.5. Determination of Phenolic Acids

The phenolic acids in the soil were extracted by an ASE-HPLC350 rapid solvent extractor (Dionex, Sunnyvale, CA, USA). A piece of cellulose film was placed at the bottom of the extraction column, and 80 g of air-dried soil was added. The soil samples were extracted with ethanol. The pressure was set to 10.3 MPa, and the temperature was set to 120 °C. The extraction time was set to 5 min, and the cycle was set to 2. The blow volume was set to 60%. Methanol was used for subsequent extraction under the same conditions. The extract solutions were steamed using a rotary evaporator at 49 °C and 190 rpm. Then, 3 mL of methanol was used to dissolve the sample, and a 0.22-μm organic filter membrane was used for filtration. Samples were analyzed using an Ultimate 3000 high-performance liquid chromatograph (Dionex, California, USA) [18]. The soil from one seedling was used as a biological replicate, and three biological replicates were analyzed for each treatment.

2.6. Determination of Soil Micro-Organisms

The dilution plating procedure was used to count the number of soil micro-organisms [19]. Bacteria were cultured in Luria–Bertani (LB) medium at 37 °C for 1 day, fungi were cultured in potato dextrose agar (PDA) medium at 28 °C for 2 days, and actinomycetes were cultured in modified Gause medium at 28 °C for 7 days. The fresh rhizosphere soil was sent to Shanghai Meiji Biomedical Technology Co., Ltd. (Shanghai, China) for the high-throughput sequencing of soil fungi.

2.7. Determination of Real-Time Fluorescence Quantitative Analysis of Soil Harmful Fungi

The DNA of soil micro-organisms was extracted using the Soil Genomic DNA Kit [20]. The SYBR Premix Ex Taq TMKit TaKaRa kit (TaKaRa Bio Inc., Kusatsu, Japan) and CFX96 TMT thermal Cycler (Bio-Rad, Hercules, CA, USA) were used to analyze the copy number of four Fusarium genes under different treatments by RT-qPCR. Three biological replicates per treatment were analyzed.

2.8. Determination of Soil Nutrients

NH4+-N: 5.00 g soil sample was mixed with 25 mL 2 mol/L KCl solution and oscillated for 1 h. After filtration, 5 mL phenol and NaClO were added into 10 mL filtrate. It was left for 1 h, and then 50 mL distilled water was added. The absorbance was measured at 625 nm. NO3−-N: 5.00 g soil sample was mixed with 50 mL 2 mol/L KCl solution and oscillated for 1 h. After standing for 5 min, the absorbance was measured at 220 and 275 nm. Available P: 2.50 g soil sample was mixed and oscillated with 50 mL NaHCO3 solution and a spoonful of non-phosphorous activated carbon for 30 min. Then, it was strained with non-phosphorous filter paper. A total of 10 mL filtrate was mixed with 35 mL distilled water and 5 mL Mo antimony colorimetric. It was incubated for 30 min, and the absorbance was measured at 700 nm. Available K: 0.20 g soil sample was mixed with 1.6 mL ammonium acetate solution, shaken, and extracted for 1 h. After centrifuging for 10 min at 10,000 r, 1 mL supernatant was mixed with 2 mL distilled water and determined with a flame photometer. Organic matter: A 0.50-gram soil sample was thoroughly mixed with a 10 mL K2Cr2O7 solution, then 20 mL of concentrated sulfuric acid was added to mix well, left for 30 min, and distilled water was added to 250 mL. The o-Phenanthroline was used as an indicator and titrated with FeSO4 standard solution. The soil from one seedling was used as a biological replicate, and three biological replicates were analyzed for each treatment.

2.9. Statistical Analysis

The statistical analysis was conducted using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Based on the operational taxonomic unit (OTU) results, the diversities of Shannon, Chao, Ace, and Simpson indices were calculated using Mothur software 1.30.2. Principal coordinate analysis (PCoA) and relative abundance of fungi community at the genus level were performed using R language (v.4.1.1).

3. Results

3.1. Antibacterial Test and Volatile Components Determination of LFP

To verify the effects of LFP on ARD, we performed the antibacterial test of LFP water solution on the main pathogens of ARD, such as Fusarium proliferatum, Fusarium solani, and Fusarium oxysporum (Figure 1). The inhibition zone of LFP on F. proliferatum, F. solani, and F. oxysporum was greater than 15 mm, which has an obvious antibacterial effect. Among them, the inhibition zone of 3% leaf fermentation product water solution was more obvious (Table 1). The diameters of the suppression circles for 3% LFP against F. proliferatum, F. solani, and F. oxysporum were 26, 24, and 30 mm, respectively. In addition, we also determined the volatile components of LFP using GC-MS, and the result showed that organic acids, phenols, and organic esters, such as dibutyl phthalate, were the main antibacterial substances (Table 2).

3.2. Effect of LFP on Growth of M. hupehensis Seedlings

The plant height, ground diameter, and fresh and dry weight of seedlings of CK2, T1, and T2 were significantly higher than those of CK1 (Figure 2). Compared with the CK1, the plant height, ground diameter, and fresh and dry weight of T1 increased by 31.9, 41.1, 67.1, and 68.7%, respectively (Table 3). Although these indicators are still lower than CK2. These indicated that the application of LFP in replanted soil can significantly promote the growth of M. hupehensis seedlings and effectively alleviate ARD.
The results of root configuration analysis showed that LFP promoted root development (Table 4). Compared with CK1, the root length, root area, root volume, and root tip number of T1 increased by 144.6, 116.4, 115.8, and 174.1%, respectively. Those of T2 increased by 55.7, 46.9, 42.1, and 89.2%, respectively. The root growth of T1 was better than that of T2. However, these indicators of T1 and T2 are lower than those of CK2.

3.3. Effects of LFP on Root Anti-Oxidant Enzymes Activity and MDA Content

Figure 3 shows that both bromomethane fumigation and LFP significantly increased root anti-oxidant oxidase activity and decreased MDA content. Compared with CK1, the activities of SOD, CAT, and POD in T1 increased by 42.0, 68.9, and 62.7%, respectively, and the content of MDA decreased by 50.0%. The effect of T1 was better than T2. And the CK2 has the best results.

3.4. Effects of LFP on the Nutrients of Soil

We determined the nutrients in the soil after the application of LFP (Table 5). Compared with CK1, T1 treatment increased the content of ammonium nitrogen, nitrate nitrogen, available phosphorus, available potassium, and organic matter by 31.8, 71.6, 36.5, 54.8, and 33.8%, respectively. The effect of T2 on increasing soil nutrients was higher than that of T1. There was no significant difference between CK2 and CK1.

3.5. Effects of LFP on the Phenolic Acids Content and Soil Enzyme Activities

We measured the difference in phenolic acid content under different treatments and found that the application of LFP effectively reduced the content of phenolic acid in soil (Table 6). Compared with CK1, the content of benzoic acid, phlorizin, phloretin, and cinnamic acid in T1 decreased by 15.9, 26.3, 23.3 and 41.1%, respectively, and those in T2 decreased by 12.9, 32.9, 26.2, and 34.2%, respectively. The soil phenolic acids content measured in CK2 was much lower than in other treatments. Contrary to the trend of phenolic acid content, soil enzyme activities showed an increasing trend in the treatment with LFP (Table 7). Compared with CK1, the activities of urease, sucrase, phosphatase, and catalase in T1 increased by 75.0, 114.8, 57.7, and 16.7%, respectively, and those in T2 increased by 37.5, 87.3, 12.3, and 11.1%, respectively. And the effect of T1 is higher than that of T2. However, there were no significant differences in soil enzyme activities between CK2 and CK1, which was much lower than T1 and T2.

3.6. Effects of LFP on the Number of Culturable Micro-Organisms and Pathogen Fungi in Soil

The number of culturable micro-organisms was determined to evaluate the number of soil micro-organisms (Table 8). The results showed that the number of bacteria in T1 increased by 214.3%, and the number of fungi decreased by 43.4% compared with CK1. Similarly, the number of bacteria in T2 increased by 223.9%, and the number of fungi decreased by 6.0%. The number of bacteria and fungi of CK2 was lower than other treatments. We also determined the biomass represented by the fungal DNA of four pathogen Fusarium in all treatments using real-time fluorescent quantitative PCR (Table 9). The results showed that the copy numbers of four Fusarium genes in the soil treated with LFP were lower than those in the replanted soil control. Compared with CK1, F. oxysporum, F. proliferatum, F. solani, and F. moniliforme in T1 decreased by 66.1, 82.7, 46.5, and 58.8%, respectively. In addition, those in T2 decreased by 57.7, 73.9, 28.3, and 52.9%, respectively. The copy numbers of four Fusarium genes of CK2 were the lowest. A total of 3% LFP (T1) exhibited higher inhibition to the Fusarium than 5% LFP (T2).

3.7. Effects of LFP on Soil Fungi Microecology

Compared with CK1, the Chao index and Ace index of soil fungi in CK2 were significantly reduced; the Shannon index and Simpson index were not significantly different from CK1. The Chao index, Ace index, and Shannon index of soil fungi in T1 were significantly higher than those in CK1, while the Simpson index was significantly lower (Figure 4a). This indicates that the application of 3% LFP (T1) significantly increased the richness and diversity of soil fungal communities.
Principal coordinate analysis (PCoA) reflected the difference and distance of microbial community structure between samples (Figure 4b). Through principal component analysis of different treatments, it can be found that the PC1 axis and PC2 axis explain 40.89 and 26.46% of the variation of microbial community structure, respectively. CK1 is in the first quadrant, CK2 is in the second quadrant, and T1 and T2 are in the fourth quadrant.
Figure 5 shows that the dominant fungi in the sample soil are Mortierella, Humicola, Pseudallescheria, Fusarium, Acremonium, and Cryptococcus. The contents of Fusarium and Humicola in CK2 were the lowest, followed by T1. Therefore, the application of LFP in replanted soil can significantly reduce the number of harmful fungi in soil and change the microbial community structure of the soil.

4. Discussion

ARD inhibits the normal growth and development of apple seedlings involving both biotic and abiotic factors in soil [21,22]. The occurrence of ARD is mainly related to the deterioration of soil conditions, such as the continuous increase of soil pathogenic fungus, the lack of soil nutrients, and the continuous accumulation of soil phenolic acid [4]. A previous study showed that a low concentration of root extract of A. sativum L. had a promotional effect on ARD, while a high concentration inhibited it. In this study, we explored the effect of different concentrations of LFP in alleviating ARD. The plant height, ground diameter, and fresh and dry weight can directly reflect the growth of the plant. Roots are the organs that absorb nutrients and water from the soil and are closely related to plant growth and development [23]. The application of LFP of A. sativum L. significantly promoted the biomass accumulation and root growth of M. hupehensis seedlings under replant conditions, which indicated that the LFP could alleviate ARD. However, the effect of a high concentration of LFP was lower than that of a low concentration, which may be due to the allelopathy of a high concentration of LFP. Allelopathy, a phenomenon in which metabolites such as phenolics secreted by plants affect the growth of other plants or themselves, is also an important reason for ARD. Methyl bromide, as a good fungicide, has excellent performance in preventing and controlling ARD and was only used as a positive control for research. However, due to its adverse impact on the environment, it is prohibited from being used in production. Although the results showed that the control effect of LFP of A. sativum L. on ARD was lower than that of methyl bromide treatment, as an environmentally friendly preparation, it was still an effective way to control ARD.
When plants are stressed, reactive oxygen species (ROS) rapidly accumulate, and excess ROS levels cause oxidative damage to cells [24,25]. Maintaining a high activity of anti-oxidant enzymes, such as SOD, POD, CAT, and the balance between ROS and anti-oxidant enzymes is an important means for plants to resist pressure [26]. Our results showed that after applying LFP, the activities of SOD, CAT, and POD in the root of M. hupehensis seedlings increased under replant conditions. The increase of anti-oxidant enzyme activities in roots ensured the effective removal of ROS. This may be the important reason that LFP can promote the normal growth of M. hupehensis under replant conditions. In addition, as a product of lipid membrane peroxidation, MDA can be used as an indicator of membrane peroxidation and plant stress resistance [27]. The MDA content in seedling root treated with LFP was lower than CK1; although it was still higher than methyl bromide treatment, it still indicated that LFP protected the integrity of the cell membrane by increasing the activity of anti-oxidant enzymes.
The deterioration of the physical and chemical properties of soils is an important manifestation of ARD. In this study, results showed that the application of LFP in replanted soil could significantly increase the content of available nitrogen, phosphorus, and potassium. LFP enrich soil organic matter content. After straw is applied to the soil, micro-organisms decompose the straw and break down the residue into organic matter [28]. At the same time, straw provides a suitable growth environment for micro-organisms and a large number of carbon sources for their reproduction [29]. A large amount of carbon is released into the soil when the straw is applied, which can promote microbial activity, enrich soil nutrients, and promote crop growth and development. Mixing biological control and straw-returning technology could effectively prevent the occurrence of diseases, increase plant height and stem thickness, improve yields, and increase market benefits. Straw mulching could also improve soil moisture and effectively increase potato yield [30]. Similar results were shown in this study, where the LFP of A. sativum L. more effectively replenished nutrients missing from replanted soil, which cannot be achieved by chemical fumigation. The increase in soil nutrient content conferred seedlings with a higher resistance to ARD.
Phenolic acids are one of the main factors causing replant disease in various crops such as apple, ginseng, and watermelon [18,31]. The excessive accumulation of phenolic acids in the soil suppresses the growth of newly planted seedlings, damages their roots, and causes replant disease [32]. Effectively reducing soil phenolic acid content is an important means to alleviate ARD. In this study, we found that the application of LFP in replanted soil could effectively reduce the content of phenolic acids. This may be ascribed to the improvement of the soil environment by the application of LFP, which increases the abundance of micro-organisms and other matter content and thus promotes the degradation of phenolic acids [33]. The decrease of phenolic acid content in soil reduced the toxic effect of excessive phenolic acid on the roots of replanted seedlings. The LFP of A. sativum L. can reduce self-toxicity by reducing the content of phenolic acids, which is an important reason to alleviate the occurrence of ARD.
Soil enzymes are products of soil microbial metabolism and plant and animal decomposition and participate in a series of biochemical reactions in soil [34]. Sucrase has the function of decomposing organic carbon, which can affect the nutrient content in soil [35]. The function of urease is to decompose organic nitrogen in the soil and convert it into inorganic nitrogen that can be directly absorbed and utilized by the plant root system, which has the effect of improving soil fertility [36]. Phosphatase breaks down phosphate fertilizer and organic phosphorus in the soil, making it available for plant uptake [37]. The function of catalase is to break down hydrogen peroxide and prevent it from causing damage to the plant root system. In this study, we found that the application of 3% LFP in replanted soil can significantly improve the activities of main soil enzymes. The increase in soil enzyme activity promoted the absorption of nutrients by seedlings, which may be partly responsible for the alleviation of ARD by LFP. It is worth noting that the soil enzyme activity of methyl bromide treatment was significantly reduced, but the control effect of ARD was still the best, indicating that the improvement of soil enzyme activity was part of the reason that LFP of A. sativum L. could control ARD, but it was not the main reason.
The increase in the number of harmful fungi can lead to ARD, which makes the soil microbial environment worse and more likely to cause the occurrence of soil-borne diseases [38]. The characteristics of the general bactericidal effect of chemical fumigants on soil micro-organisms indicate that micro-organisms are the main cause of ARD, and chemical fumigation is an effective way to prevent and control ARD. However, most chemical fumigants have been gradually abandoned due to their adverse effects on the environment. In this study, methyl bromide treatment showed the best effect against ARD and was used as a positive control. The application of straw into the soil provided favorable conditions for microbial reproduction and increased the number and diversity of micro-organisms. Numerous studies have shown that applying straw to replanted soils could enrich nutrients in the soil, prevent nutrient imbalance, and improve the structure of microbial communities [39]. This may be due to the substances produced by straw decomposition inhibiting the growth of harmful fungi while providing a favorable environment for other micro-organisms and accelerating the reproduction of beneficial micro-organisms. Previous studies found that the application of leaf decomposition in soil produced some chemosensory substances, which had different effects on different micro-organisms [40]. Similar results were found in our study. The number of culturable fungi and the abundance of Fusarium were inhibited after the application of LFP, while the number of bacteria increased. In addition to inhibiting soil harmful fungi, LFP of A. sativum L. are more inclined to balance soil microbial community structure, which is an important difference from chemical fumigants. Furthermore, different concentrations of leaf fermentation had different effects on soil community structure [41]. It is necessary to consider the concentration of LFP in the control of ARD. In this study, the application of LFP could reduce the number and abundance of harmful fungi in the soil, regulate the microbial community structure of replanted soil, and promote the transformation of replanted soil from “fungal type” to “bacterial type”. A total of 3% of LFP had the best effect. However, the reason for LFP of A. sativum L. changing the structure of microbial communities needs further study.
In summary, we found that the application of LFP of A. sativum L. in replanted soil could promote the growth of M. hupehensis seedlings and increase the root anti-oxidant oxidase enzyme activities. In addition, LFP improved soil enzyme activity and soil nutrition, reduced phenolic acid content, increased the number of culturable bacteria, and decreased the number of harmful fungi, which optimized the soil structure and microbial environment. These findings provide evidence that the LFP of A. sativum L. are effective in alleviating ARD, with a 3% concentration being the most effective, which is of great significance for the utilization of dry leaves of A. sativum L. and the prevention and control of ARD.

Author Contributions

Conceptualization, Z.M. and C.Y. (Chengmiao Yin); methodology, Y.Z.; software, Y.Z.; validation, C.Y. (Chengwen Yin), R.Z., G.W. and Y.L.; investigation, C.Y. (Chengwen Yin) and G.W.; data curation, C.Y. (Chengwen Yin) and Y.Z.; writing—original draft preparation, R.Z. and Y.L.; writing—review and editing, C.Y. (Chengmiao Yin) and Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the China Agriculture Research System of MOF and MARA (CARS-27), the National Natural Science Foundation of China (Grant No. 31672104), the Taishan Scholar Funded Project (No. ts20190923), the Key R&D Program of Shandong Province (2022TZXD0037).

Data Availability Statement

All the data are contained within the article.

Acknowledgments

We are grateful to Zhijun Zhang (Northwest A&F University), Linjiao Sun (Shandong Agricultural University), Xiaolong Zhou (Shandong Agricultural University), and Yi Lv (Weihai Academy of Agricultural Sciences) for their help in the completion of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Inhibitory effects of LFP on Fusarium proliferatum (a), Fusarium solani (b), Fusarium oxysporum (c). The hole in the red circle is the LFP of A. sativum L. at 3% concentration.
Figure 1. Inhibitory effects of LFP on Fusarium proliferatum (a), Fusarium solani (b), Fusarium oxysporum (c). The hole in the red circle is the LFP of A. sativum L. at 3% concentration.
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Figure 2. Effects of LFP on Malus hupeheusis Rehd. seedings. CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP.
Figure 2. Effects of LFP on Malus hupeheusis Rehd. seedings. CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP.
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Figure 3. Effects of LFP on root anti-oxidant oxidase enzyme activities and MDA content of M. hupehensis seedling. (a) Root CAT activity under different treatments; (b) Root SOD activity under different treatments; (c) Root POD activity under different treatments; (d) Root MDA content under different treatments. Different letters indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
Figure 3. Effects of LFP on root anti-oxidant oxidase enzyme activities and MDA content of M. hupehensis seedling. (a) Root CAT activity under different treatments; (b) Root SOD activity under different treatments; (c) Root POD activity under different treatments; (d) Root MDA content under different treatments. Different letters indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
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Figure 4. Alpha diversity and PCoA analysis of soil fungal community. (a) Simpson index, Ace index, Shannon index, and Chao index of OTU level under different treatment. (b) PCoA analysis of soil fungal communities under different treatments. CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP.
Figure 4. Alpha diversity and PCoA analysis of soil fungal community. (a) Simpson index, Ace index, Shannon index, and Chao index of OTU level under different treatment. (b) PCoA analysis of soil fungal communities under different treatments. CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP.
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Figure 5. Relative abundance and significant difference of soil fungal community under different treatments. (a) Fungal community abundance percentages at the genus level. (b) Kruskal–Wallis H test at the genus level. CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP.
Figure 5. Relative abundance and significant difference of soil fungal community under different treatments. (a) Fungal community abundance percentages at the genus level. (b) Kruskal–Wallis H test at the genus level. CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP.
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Table 1. Inhibitory effects of LFP on Fusarium.
Table 1. Inhibitory effects of LFP on Fusarium.
Concentration of Water Solution of LFP (%)Fusarium proliferatumFusarium solaniFusarium oxysporum
Bacteriostatic Zone Diameter (mm)
0---
3262430
5182016
Table 2. Analysis of volatile components of LFP.
Table 2. Analysis of volatile components of LFP.
NameMolecular FormulaRelative ContentCAS
Valeric acidC5H10O216.06109-52-4
2-Chloroethyl vinyl etherC4H7ClO8.97110-75-8
3-Ethoxy-1-propanolC7H18O34.76111-35-3
Hexanoic acidC6H12O24.09142-62-1
Butyric acidC4H8O22.51107-92-6
Hydrocinnamic acidC9H10O22.4501-52-0
Dibutyl phthalateC16H22O41.8284-74-2
Methyl valerateC6H12O21.09624-24-8
Isoamyl isovalerateC10H20O21.07659-70-1
Table 3. Effects of LFP on plant biomass of Malus hupeheusis Rehd. seedings.
Table 3. Effects of LFP on plant biomass of Malus hupeheusis Rehd. seedings.
TreatmentPlant Height (cm)Ground Diameter (mm)Fresh Weight (g)Dry Weight (g)
CK158.33 ± 3.72 d6.98 ± 0.15 c62.66 ± 4.39 c24.97 ± 1.79 c
CK285.33 ± 4.20 a10.13 ± 0.15 a114.52 ± 8.95 a45.67 ± 3.68 a
T176.93 ± 3.40 b9.85 ± 0.08 b104.71 ± 3.12 b42.13 ± 1.39 b
T261.27 ± 3.72 c8.21 ± 0.09 bc63.67 ± 2.69 c25.47 ± 0.82 c
Note: CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP. Different letters in the same column indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
Table 4. Effects of LFP on root parameters of M. hupehensis seedlings.
Table 4. Effects of LFP on root parameters of M. hupehensis seedlings.
TreatmentLength (cm)Surface Area (cm2)Volume (cm3)Tips
CK1832.00 ± 23.21 d384.33 ± 14.05 d7.64 ± 0.29 d2322.67 ± 105.53 d
CK22360.67 ± 56.70 a937.37 ± 65.34 a18.71 ± 0.38 a7398.00 ± 251.81 a
T12034.67 ± 64.84 b831.01 ± 69.91 b16.49 ± 0.55 b6364.33 ± 170.94 b
T21295.67 ± 54.15 c564.65 ± 18.85 c10.86 ± 0.37 e4393.33 ± 212.80 c
Note: CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP. Different letters in the same column indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
Table 5. Effects of LFP on soil nutrients.
Table 5. Effects of LFP on soil nutrients.
TreatmentNH4+-N
(mg·kg−1)		NO3−-N
(mg·kg−1)		Available P
(mg·kg−1)		Available K
(mg·kg−1)		Organic Matter
(g·kg−1)
CK13.18 ± 0.06 b6.98 ± 0.30 b26.60 ± 1.27 b69.05 ± 1.99 b7.45 ± 0.17 b
CK23.17 ± 0.06 b7.17 ± 0.31 b26.18 ± 0.53 b69.05 ± 5.26 b7.61 ± 0.11 b
T14.19 ± 0.23 a11.98 ± 0.17 a36.31 ± 0.71 a106.94 ± 1.99 a9.97 ± 0.12 a
T24.06 ± 0.21 a12.31 ± 0.14 a36.55 ± 0.28 a114.97 ± 1.99 a10.32 ± 0.07 a
Note: CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP. Different letters in the same column indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
Table 6. Effects of LFP on the content of phenolic acids in soil.
Table 6. Effects of LFP on the content of phenolic acids in soil.
TreatmentBenzoic Acid
(mg·kg−1)		Phloridzin
(mg·kg−1)		Phloretin
(mg·kg−1)		Cinnamic Acid
(mg·kg−1)
CK16.58 ± 0.19 a2.13 ± 0.12 a1.72 ± 0.11 a0.73 ± 0.02 a
CK23.13 ± 0.03 c1.02 ± 0.04 d1.15 ± 0.02 c0.37 ± 0.03 c
T15.53 ± 0.12 bc1.57 ± 0.10 b1.32 ± 0.03 b0.43 ± 0.03 bc
T25.73 ± 0.12 b1.43 ± 0.07 c1.27 ± 0.05 bc0.48 ± 0.02 b
Note: CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP. Different letters in the same column indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
Table 7. Effects of LFP on soil enzyme activity.
Table 7. Effects of LFP on soil enzyme activity.
TreatmentUrease Activity
(mg·g−1·d−1)		Sucrase Activity
(mg·g−1·d−1)		Phosphatase Activity
(mg·g−1·d−1)		Catalase Activity
(mg·g−1·d−1)
CK10.08 ± 0.00 c7.31 ± 0.11 bc11.25 ± 0.74 bc0.18 ± 0.01 c
CK20.08 ± 0.00 c6.72 ± 0.24 c8.69 ± 0.34 c0.18 ± 0.00 c
T10.14 ± 0.00 a15.70 ± 0.45 a17.74 ± 0.61 a0.21 ± 0.01 a
T20.11 ± 0.01 b13.69 ± 0.62 b12.63 ± 0.51 b0.20 ± 0.01 b
Note: CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP. Different letters in the same column indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
Table 8. Effects of LFP on the number of soil culturable micro-organisms.
Table 8. Effects of LFP on the number of soil culturable micro-organisms.
TreatmentBacteria (×105·CFU·g−1)Fungi (×103·CFU·g−1)Bacteria/Fungi
CK17.00 ± 1.00 b27.67 ± 5.03 a25.30 d
CK22.33 ± 1.15 c4.00 ± 1.00 c58.25 c
T122.00 ± 1.00 a15.67 ± 1.53 b140.39 a
T222.67 ± 1.155 a26.00 ± 1.00 ab87.19 b
Note: CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP. Different letters in the same column indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
Table 9. Real-time qPCR analysis of Fusarium under different treatments.
Table 9. Real-time qPCR analysis of Fusarium under different treatments.
TreatmentGene Copies
F. oxysporum
(109·Copies·g−1)		F. proliferatum
(106·Copies·g−1)		F. solani
(1011·Copies·g−1)		F. moniliforme
(106·Copies·g−1)
CK112.21 ± 1.23 a18.72 ± 1.72 a2.26 ± 0.16 a7.28 ± 0.36 a
CK21.27 ± 0.06 d2.47 ± 0.19 d0.32 ± 0.02 d1.62 ± 0.12 d
T14.14 ± 0.35 c3.23 ± 0.14 c1.21 ± 0.05 c3.00 ± 0.26 c
T25.16 ± 0.22 b4.88 ± 0.31 b1.62 ± 0.17 b3.43 ± 0.27 b
Note: CK1: untreated control soil; CK2: methyl bromide fumigation; T1: 3% of the soil mass of LFP; T2: 5% of the soil mass of LFP. Different letters in the same column indicate significant differences between treatments based on Tukey’s multiple range test (p < 0.05).
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MDPI and ACS Style

Yin, C.; Zhang, R.; Zhao, Y.; Wang, G.; Yin, C.; Liu, Y.; Mao, Z. Leaf Fermentation Products of Allium sativum L. Can Alleviate Apple Replant Disease (ARD). Horticulturae 2024, 10, 546. https://doi.org/10.3390/horticulturae10060546

AMA Style

Yin C, Zhang R, Zhao Y, Wang G, Yin C, Liu Y, Mao Z. Leaf Fermentation Products of Allium sativum L. Can Alleviate Apple Replant Disease (ARD). Horticulturae. 2024; 10(6):546. https://doi.org/10.3390/horticulturae10060546

Chicago/Turabian Style

Yin, Chengwen, Rong Zhang, Yiming Zhao, Gongshuai Wang, Chengmiao Yin, Yusong Liu, and Zhiquan Mao. 2024. "Leaf Fermentation Products of Allium sativum L. Can Alleviate Apple Replant Disease (ARD)" Horticulturae 10, no. 6: 546. https://doi.org/10.3390/horticulturae10060546

APA Style

Yin, C., Zhang, R., Zhao, Y., Wang, G., Yin, C., Liu, Y., & Mao, Z. (2024). Leaf Fermentation Products of Allium sativum L. Can Alleviate Apple Replant Disease (ARD). Horticulturae, 10(6), 546. https://doi.org/10.3390/horticulturae10060546

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