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The Endophytic Bacteria Bacillus velezensis Lle-9, Isolated from Lilium leucanthum, Harbors Antifungal Activity and Plant Growth-Promoting Effects

Bacillus velezensis is an important plant growth-promoting rhizobacterium with immense potential in agriculture development. In the present study, Bacillus velezensis Lle-9 was isolated from the bulbs of Lilium leucanthum. The isolated strain showed antifungal activities against plant pathogens like Botryosphaeria dothidea, Fusarium oxysporum, Botrytis cinerea and Fusarium fujikuroi. The highest percentage of growth inhibition i.e., 68.56±2.35% was observed against Fusarium oxysporum followed by 63.12 ± 2.83%, 61.67 ± 3.39% and 55.82 ± 2.76% against Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi, respectively. The ethyl acetate fraction revealed a number of bioactive compounds and several were identified as antimicrobial agents such as diketopiperazines, cyclo-peptides, linear peptides, latrunculin A, 5α-hydroxy-6-ketocholesterol, (R)-S-lactoylglutathione, triamterene, rubiadin, moxifloxacin, 9-hydroxy-5Z,7E,11Z,14Zeicosatetraenoic acid, D-erythro-C18-Sphingosine, citrinin, and 2- arachidonoyllysophosphatidylcholine. The presence of these antimicrobial compounds in the bacterial culture might have contributed to the antifungal activities of the isolated B. velezensis Lle- 9. The strain showed plant growth-promoting traits such as production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, and nitrogen fixation and phosphate solubilization. IAA production was accelerated with application of exogenous tryptophan concentrations in the medium. Further, the lily plants upon inoculation with Lle-9 exhibited improved vegetative growth, more flowering shoots and longer roots than control plants under greenhouse condition. The isolated B. velezensis strain Lle-9 possessed broad-spectrum antifungal activities and multiple plant growth-promoting traits and thus may play an important role in promoting sustainable agriculture. This strain could be developed and applied in field experiments in order to promote plant growth and control disease pathogens.

Organic Acid Production Assay
Organic acids in the isolated strain Lle-9 were detected according to the protocol developed by Cunningham and Kuiack [30]. About 50 μl of the bacterial suspension in MgSO 4 (10 mM) was inoculated in 800 μl of Sucrose Tryptone medium (ST) containing 20 g l −1 sucrose and 5 g l −1 tryptone. The ST medium was supplemented with 10 ml of trace element solution. Samples were incubated at 30°C and 200 rpm shaking for 5 days. After incubation, organic acids in samples were detected by adding 100 μl of 0.1% alizarine red S pH indicator. After 15 min, samples with yellow color were considered as positive. Pink-colored samples indicated negative results.

Indole Acetic Acid (IAA) Detection
Indole acetic acid (IAA) production in the isolated strain Lle-9 was assayed according to the method of Gordon and Weber [31]. A bacterial suspension of 150 μl prepared in MgSO 4 (10 mM) was inoculated in 3 ml of 1/10 diluted 869-rich medium. The medium was supplemented with various tryptophan concentrations of 0 mg ml -1 , 2 mg ml -1 , 4 mg ml -1 , and 6 mg ml -1 . Samples were incubated at 30 o C for 4 days with 150-180 rpm shaking in the dark. After incubation, the cultures were centrifuged at 4,000 ×g for 20 min. About 1 ml of the supernatant was mixed with 2 ml of Salkowski's reagent (98 ml 35% HClO 4 , 2 ml 0.5M FeCl 3 ). After 20 min, change of color from yellow to pink was considered as positive for IAA production. Quantitative measurement of indole acetic acid in the samples was conducted by measuring OD at 530 nm in a spectrophotometer. The IAA quantities in samples were measured based on a standard curve of known values (Fig. S1).

Siderophore Detection
The siderophore production in the strain Lle-9 was evaluated through both qualitative and quantitative tests. The bacterial cells were cultured in liquid 284 medium with chrome azurol S (CAS) shuttle solution, according to the described method [32]. About 50 μl of the bacterial suspension in 10 mM MgSO 4 was added to 800 μl of 284 medium prepared with three different iron concentrations. The iron concentrations used were: 0 μM, 0.25 μM, and 3 μM Fe(III) citrate. Samples were incubated at 30°C for 5 days with 150 rpm shaking. After incubation, 100 μl of the blue Chromium Azurol S (CAS) reagent was added to samples followed by incubation for 4 h at room temperature. After incubation, the change of color from blue to orange/yellow was considered as positive. Siderophore concentrations in all samples were further measured at 630 nm. The siderophore quantities were measured as % of siderophore units by the formula: % of siderophore units = Ar − As/Ar * 100 where, "Ar" is the absorbance of reference (CAS reagent); and "As" is the absorbance of sample at 630 nm. The ability of the strain to produce siderophores was further confirmed through qualitative test using CAS agar assay. All assays were carried out in triplicates.

Experimental Design of Greenhouse Test
The growth-promoting effects of the isolated endophytic bacterial strain Lle-9 were evaluated on the Asiatic lilium hybrids 'Tresor. ' Same-sized bulbs with normal and healthy appearance were selected from the storage house at 4 o C. For inoculation, the isolated strain Lle-9 was cultured in 5 ml LB for 10-15 h followed by further inoculation in 50 ml LB for 24 h at 30 o C with 220 rpm shaking. After incubation, the culture was re-inoculated in 400 ml LB and was kept to grow at 30 o C for 24 h. This culture was then diluted 10 times with normal water and bulbs of Tresor variety were soaked in the diluted culture for 40 min. The non-inoculated bulbs, soaked in simple LB, were used as controls. Soil pots of sizes 20 × 30 cm were prepared with soil mix of peat moss, perlite, and vermiculite in a ratio of 2:1:1. Three lily bulbs, either inoculated or non-inoculated control were sown in each soil pot. Pots were kept in a completely randomized design (CRD). Each treatment contained 5 pots. Pots were kept in plastic trays with holes in the bottom. The plastic trays were watered with equal amounts of normal tap water at regular intervals. Morphological data such as number of flowering shoots, plant height, leaf length, leaf width, bulb size and weight and root length were taken at the peak vegetative and reproductive stage.

Statistical Analysis
The data obtained were subjected to analysis of variance (ANOVA). Means were compared with Student's t-test at a probability of α = 0.05.

Isolation and Identification of B. velezensis Strain Lle-9
In our study, several bacterial endophytes were isolated from the bulb samples of Lilium leucanthum. Based on the antifungal assays, the isolated endophytic strain Lle-9 of B. velezensis was selected for further assays. The isolated strain Lle-9 formed off whitish chalky colonies with internal whitish circular lines (Fig. 1A), as a grampositive and spore-forming bacterium and exhibited small rod-shaped structures typical of the genus Bacillus as revealed by Scanning Electron Microscopic (SEM) analysis (Figs. 1B and 1C).
Molecular analysis indicated that the isolated strain Lle-9 belongs to the genus Bacillus. The BLAST results revealed that the 16S rRNA gene sequence was closely related to Bacillus velezensis. Based on the maximum likelihhod phylogenetic tree constructed with the 16S rRNA similarity (%), the Lle-9 strain revealed 99.34% similarity with Bacillus velezensis strain CR-502 (T), (AY603658) (Fig. 2). The 16S rRNA gene sequence of the isolated strain shared high similarities with other Bacillus species such as 99.25% with Bacillus siamensis, 99.25% with Bacillus nakamurai, and 99.16% with both Bacillus amyloliquefaciens and Bacillus subtillus. The 16S rRNA gene sequence of the isolated strain Lle-9 was submitted to GenBank under accession number MN461530.1.

Antifungal Activity Analysis
The isolated endophytic strain Lle-9 showed high potential of broad-spectrum antifungal activities against the tested phytopathogens, i.e., Botryosphaeria dothidea, Fusarium oxysporum, Fusarium fujikuroi, and Botrytis cinerea (Fig. 3A). These pathogenic strains were previously tested in an in vitro study for their pathogenicity potential and disease-causing ability in bulbs of Asiatic lilium hybrid 'Tresor. ' All pathogenic fungal strains revealed disease symptoms in bulbs of Tresor (Fig. S2). The strain Lle-9 exhibited considerable inhibition potential as revealed by zones of inhibition of the test pathogens on PDA plates. These high activities might be due to the release of some diffusible compound(s) against the test pathogens. Zones of inhibition of pathogenic fungi on PDA plates were measured as percentage values. The highest percentage of growth inhibition, i.e., 68.56 ± 2.35%, was observed against Fusarium oxysporum followed by 63.12 ± 2.83%, 61.67 ± 3.39%, and 55.82 ± 2.76% against Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi, respectively (Fig. 3B).

Plant Growth-Promoting (PGP) Assays
The plant growth-promoting effects of Lle-9 were assayed both qualitatively and quantitatively. According to our results, the endophytic bacterial strain Lle-9 showed positive results for all conducted assays.

ACC (Deaminase) and Organic Acid Detection
The ACC deaminase production was detected through a qualitative test based on change of color. The strain Lle-9 was found positive for the production of ACC deaminase (Table 1 and Fig. 4A).
The isolated strain Lle-9 was assayed for production of organic acids through a qualitative test. Strain Lle-9 showed moderate to high production of organic acids as revealed by the change of color from pink to yellow (Table 1 and Figs. 4A and 4B).

Indole Acetic Acid
Indole acetic acid (IAA) production in the isolated strain Lle-9 was detected through both qualitative and quantitative tests. Qualitative test confirmed IAA production in Lle-9 as revealed by the change of color of the culture supernatant from yellow to pink (Figs. 4A and 4C). Further, IAA was quantified in the strain at various tryptophan concentrations supplemented in the culture medium. The strain Lle-9 was able to produce IAA at different tryptophan concentrations (Table 1). Different tryptophan concentrations impacted the IAA production in the strain. The IAA content in the isolated strain increased with increasing tryptophan in the culture medium. The strain Lle-9 showed lower IAA content, i.e., 23.2 ± 1.9 μg ml -1 , at tryptophan concentration of 0 mg ml -1 . However, the IAA content increased gradually with increasing the tryptophan concentrations from 0 mg ml -1 to  6 mg ml -1 in the culture medium. The endophytic strain Lle-9 produced 79.7 ± 3.3 μg ml -1 , 117.7 ± 3.5 μg ml -1 , and 165.7 ± 5.8 μg ml -1 at tryptophan concentrations of 2 mg ml -1 , 4 mg ml -1 , and 6 mg ml -1 in the culture medium, respectively. These results suggest that the isolated strain Lle-9 has the potential to produce high content of indole acetic acid even in the absence of exogenous tryptophan. Application of exogenous tryptophan had no negative impact on IAA production; rather, it increased the production and a positive correlation was observed between the IAA production and the increasing tryptophan concentrations.

Siderophores
Production of siderophores in Lle-9 was assayed both qualitatively and quantitatively at different Fe(III) citrate concentrations added to the culture medium. The endophytic strain Lle-9 was able to produce siderophores as confirmed through a change of color from blue to orange yellow (Figs. 4A and 4D). To further test whether an iron source has any impact on the production of siderophores, the strain was cultured in the liquid 284 medium supplemented with different Fe(III) citrate concentrations ( Table 1). The strain Lle-9 showed high siderophore production when cultured in medium without Fe(III) citrate. The total siderophore quantity was reported as 51.3 ± 3.8 (psu) in the culture medium without addition of Fe(III) citrate. However, the siderophore accumulation by the strain declined as the quantities of Fe(III) citrate increased in the culture medium. Significantly greater decrease in siderophores was observed when Fe(III) citrate increased from 0 μM to 0.25 μM. About 33.3 ± 1.5 (psu) siderophores were detected at 0.25 μM Fe(III) citrate in the medium. Further reduction in siderophores was observed when Fe(III) citrate concentration was increased to 3.0 μM. However, this was not significantly different from the quantities obtained at 0.25 μM. At 3.0 μM Fe(III) citrate concentration, the strain Lle-9 accumulated 30.1 ± 1.3 (psu) siderophores. The siderophore production in the endophytic strain Lle-9 was further assayed through a qualitative test using chrome azurol S (CAS) on agar plates. An orange/yellow hallow was observed around the colonies of Lle-9 indicating the production of siderophores. The strain Lle-9 was able to quench the iron from the dye complex that resulted in a change of color from blue to orange/yellow in the form of a hallow surrounding the bacterial colony (Fig. 4B). Further, the diameter of the yellow/orange hallow produced by the strain averaged 15.32 ± 1.3 mm. This test further confirmed the high potential of the isolated endophytic strain Lle-9 to produce siderophores.

Potential for Nitrogen Fixation and Phosphate Solubilization
The nitrogen-fixing potential of the isolated B. velezensis Lle-9 was assessed by its ability to grow on nitrogenfree minimal medium (NFM). The Escherichia coli strain DH5α, which is unable to grow on nitrogen-free medium was used as a negative control. Both strains were cultured on nitrogen-free medium and medium supplemented with 5 mM NH 4 Cl, which is a preferred source of nitrogen. Results revealed that the growth of B. velezensis Lle-9 was clearly visible on the NFM, while the Escherichia coli DH5α, which does not fix nitrogen, grew only on medium supplemented with reactive nitrogen (Fig. 4C). These results demonstrate the potential of the isolated endophytic B. velezensis Lle-9 to fix nitrogen.
The phosphate solubilization potential of the endophytic B. velezensis strain Lle-9 was assayed on solid NBRIP medium. In this medium, Ca 3 (PO 4 ) 2 was provided as the sole source of inorganic phosphate. The endophytic strain Lle-9 was able to grow on the medium for longer incubation time and it solubilized the inorganic phosphate as indicated by the clearing zone surrounding bacterial colonies (Fig. 4D).

Plant Growth Promotion
The plant growth-promoting potential of the isolated B. velezensis Lle-9 was further assessed on the vegetative growth and bulbs production of the Asiatic lilium hybrids 'Tresor' under greenhouse conditions. Bulbs of 'Tresor' were inoculated with the isolate strain before cultivation in soil pots. Upon completion of vegetative growth, a number of plant growth parameters like plant height, number of flowering shoots, leaf length, leaf width, stem diameter and weight of bulbs were measured between the inoculated and un-inoculated control plants. Our results revealed that inoculation of bulbs with B. velezensis Lle-9 resulted in significant increase in overall plant growth relative to control plants ( Table 2). Inoculation of bulbs led to a significant increase in most of the tested growth parameters. The inoculated plants showed significantly high (p ≤ 0.05) increase in the number of flowering shoots, i.e. 3.47 ± 0.30, as compared to 2.73 ± 0.45 in un-inoculated control plants. Likewise, significant differences were observed in plant height between inoculated and un-inoculated plants (Fig. 5A, Table 2 (Fig. 5B). Overall, these results revealed that the B. velezensis Lle-9 was able to improve the vegetative and reproductive growth of lily plants upon inoculation.

Discussion
In the present study, a new endophytic bacterial strain Lle-9 of Bacillus velezensis was isolated from the bulbs of Lilium leucanthum. The isolated strain was assessed for its potential of antifungal activities against several fungal pathogens and it showed antimicrobial activity and resisted the growth and proliferation of these pathogens. The strain was identified to be B. velezensis through morphological and molecular analysis. Several plant growth promotion assays were conducted to confirm whether the strain could promote growth of the associated plants through several underlined mechanisms and whether it could also provide protection against disease-causing agents. Previous studies indicated that several B. velezensis strains isolated from various hosts, showed broadspectrum antimicrobial activities and plant growth promotion effects. Bacillus amyloliquefaciens FZB42, now recognized as a B. velezensis strain was reported in 2007 as the first gram-positive biocontrol bacteria to have its genome sequenced [36]. This isolate is now used as a model strain to promote plant growth and to confer disease resistance against broad-spectrum phytopathogens [37]. In addition, several other strains of B. velezensis have been used as antagonists of plant pathogens and as plant growth promoters in sustainable agriculture [12,36,38]. Further, a Bacillus velezensis strain CC09, isolated from healthy leaves of Cinnamomum camphora, showed immense potential as a new biocontrol agent, in control of many phytopathogenic diseases including wheat powdery mildew disease [39]. A B. velezensis strain NJAU-Z9, isolated from pepper rhizosphere, showed growth promotion effects in pepper [40].
In the present study, the isolated strain Lle-9 showed considerable antagonistic effects against fungal phytopathogens like Fusarium oxysporum, Botryosphaeria dothidea, Botrytis cinerea, and Fusarium fujikuroi. These pathogens have previously been reported to cause serious diseases in several crop plants [41]. These phytopathogens may cause diseases in Lilium species as revealed by a test confirming pathogenicity potential against cultivated species/varieties like Lilium davidii and Tresor. One of these phytopathogens, Fusarium fujikuroi, was isolated and identified from in vitro bulbs of Lilium wardii, a Lilium species. This could be a diseasecausing agent in Lilum wardii and other Lilum species. Interestingly, the isolated endophytic strain Lle-9 exhibited higher antifungal activities against all tested fungal pathogens and was found very effective against growth and proliferation of Fusarium fujikuroi.
The ability of the strain Lle-9 to suppress the growth of phytopathogens could be due to the presence of compounds and metabolites with antimicrobial properties. To provide evidence of this, the ethyl acetate fraction was assessed for potential secondary metabolites with bio-control properties. A number of secondary metabolites were identified and showed close homologies with the already known compounds with bio-control properties. Some of the prominent compounds and group of compounds, putatively identified from the isolated strain were diketopiperazines, cyclo-peptides, linear peptides, latrunculin A, 5alpha-hydroxy-6-ketocholesterol, (R)-Slactoylglutathione, triamterene, rubiadin, moxifloxacin, 9-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid, Derythro-C18-sphingosine, citrinin, and 2-arachidonoyllysophosphatidylcholine. Isolation of these secondary metabolites and compounds is evidence that the Lle-9 has a high potential of restricting the growth and proliferation of disease-causing fungal pathogens. Our results are supported by previous identification of these bio-control compounds and metabolites from other Bacillus species. Diketopiperazines and linear di-and tripeptides were previously recovered from cultures of endophytic bacterial isolates. Syed-Ab-Rahman et al. [42] found diketopiperazines in the cultures of Bacillus amyloliquefaciens, Bacillus velezensis, and Acinetobacter sp. They further reported that the presence of these bioactive compounds resulted in resistance to phytophthora infection and plant growth promotion. A number of other studies also reported isolation of diketopiperazines (cyclo di-peptides) that resulted into broad-spectrum antimicrobial activities [43,44]. Cyclo (Leu-Leu) found in Lactobacillus plantarum AF1 isolated from kimchi was found with antifungal activity against Aspergillus flavus [45]. Cyclo (Pro-Phe) in Bacillus amyloliquefaciens Q-426 was found to have a significant antifungal activity [46]. In addition, dipeptides and tripeptides have also been proved effective in conferring fungal resistance [47]. In the present study, latrunculin A was detected in the Lle-9 extract. This compound was previously shown as having fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae [48]. Triamterene, found in the ethyl acetate fraction of Lle-9 was previously found in ayurvedic medicine, Salmali niryasa, from a medicinal plant, Bombax ceiba [49]. The salmali resin had shown strong antimicrobial and antioxidant activities. Rubiadin, a bioactive compound detected in the present study was isolated from the roots of Morinda elliptica L. (Rubiaceae) [50]. This compound demonstrated anti-HIV, cytotoxic and antimicrobial activities [51]. Recently, rubiadin (AQ1) and rubiadin 1-methyl ether (AQ2), two photosensitizing anthraquinones (AQs) isolated from Heterophyllaea pustulata, showed reduction in biofilms formation of Candida tropicalis, a common cause of fungal infections [52]. Moxifloxacin was another important bioactive compound detected in the present study. This compound was previously shown as having antimicrobial effects [53]. The presence of these bioactive compounds might be responsible for the significant antifungal activities of Lle-9 against the tested fungal pathogens.
Previous studies have revealed that the plant growth-promoting traits in isolated strains of Bacillus species were correlated with several mechanisms such as production of organic acids, siderophores, lowering plant ethylene levels by ACC deaminase production, synthesis of plant growth-regulating hormones like indole-acetic acid and cytokinins, nitrogen fixation and phosphate solubilization [54]. In the present study, we confirmed through various tests that the isolated strain Lle-9 possessed plant growth-promoting traits. The isolated strain Lle-9 was capable of producing organic acids as revealed by a qualitative test. Organic acids play an important role in plant growth promotion and defense against phytopathogens [55].
Moreover, production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase is one of the important characteristics of plant growth-promoting microbes and endophytes. ACC deaminase cleaves ACC, the immediate precursor of the plant hormone ethylene, to produce α-ketobutyrate and ammonia [56]. Ethylene serves as an important signaling molecule in plants under biotic and abiotic stresses and results in plant growth inhibition [57]. Previous studies have reported that inoculation of plants with ACC deaminase-producing microbes decreased ethylene levels that resulted in decreased inhibition of plant growth under biotic and abiotic stresses [3,58]. Previous studies showed that improvement of several crops, inoculated with Bacillus species, might partly be due to the production of ACC deaminase. The B. velezensis strain BACO 3 was reported to produce ACC deaminase, indole acetic acid, and ammonia, resulting in improved plant growth in terms of high biomass of leaves and roots in radish [59]. Bacillus species isolated from seeds of commercial tomato varieties exhibited multiple plant growth-promoting traits such as production of ACC deaminase, IAA, siderophores, and potential of nitrogen fixation and phosphate solubilization [60]. One of the isolates, HYT-12-1, identified as Bacillus subtilis, showed the highest ACC deaminase activity, which resulted in growth enhancement of tomato seedlings under greenhouse conditions. Consistent with the mentioned reports, the isolated strain Lle-9 in the present study exhibited ACC deaminase activity that might partly contributed to the improvement of growth of lily varieties upon inoculation.
Indole acitic acid is one of the important auxins that directly support plant growth and productivity. The ability of PGPRs including Bacillus species is partly attributed to their potential of IAA production that directly promotes growth of associated plants. In this connection, the role of the tryptophan precursor of IAA is important as considerable amounts of IAA are produced in the presence of excess tryptophan [61]. In the present study, the exogenous tryptophan concentrations had no negative impact on IAA production in Lle-9 and a positive correlation was observed between the tryptophan concentrations and IAA production. It seems the isolated strain Lle-9 produced IAA in a tryptophan-dependent pathway. Our results are supported by previous reports where application of exogenous tryptophan enhanced the IAA production. The Bacillus amyloliquefaciens strain FZB42, now a B. velezensis strain, was reported with production of IAA and its capacity increased five-fold with application of exogenous tryptophan [62]. Bacillus thuringiensis and Bacillus cereus isolated from soil samples exhibited IAA production in the absence and presence of L-tryptophan [63]. It was reported that the IAA production in the isolates increased with increasing concentrations of exogenous tryptophan.
Iron is an essential element necessary for growth of plants and microorganisms. However, it is abundantly present in soil in the form of insoluble Fe 3+ oxy-hydroxides. Plant-associated microbes reduce Fe 3+ to Fe 2+ with the help of ferrireductases or solubilize it with extracellular Fe 3+ chelators called 'siderophores' [64]. These soluble Fe 3+ -siderophore complexes are then available to both plants and microbes. Species of the genus Bacillus were previously reported with production of siderophores. Chen et al. (2007) reported siderophore bacillibactin production in B. amyloliquefaciens FZB42, now B. velezensis, and described that the siderophore synthesis in the strain was catalyzed by nonribosomal peptide synthetases. They further reported that production of high concentrations of siderophore bacillibactin in the FZB42 strain inhibited the growth of phytopathogenic bacterial and fungal competitors by depriving them of essential iron ions. Some recent studies also reported production of siderophores in Bacillus species [65,66]. Kesaulya et al. [65] reported siderophore production in the isolated Bacillus sp. from potato rhizospheric soil. They further reported that the isolated strain inhibited the pathogen causing banana wilt disease. These reports reveal that siderophore production by the plant-associated microbes not only helps plants acquire access to limited iron supply in the soil but also confers plants with a selective advantage over the pathogenic microbes by depriving them of essential iron that leads to disease resistance. In the present study, the high siderophore production in the isolated strain Lle-9 might partly be responsible for considerable antagonistic effects against the tested pathogenic strains. Quantitative evaluation of siderophores was conducted under different iron concentrations. Maximum siderophore production was observed in the absence of Fe(III) citrate in the medium. However, increasing iron concentrations in the medium resulted in reduction in siderophore accumulation. These results are supported by previous reports where an inverse relationship was observed between siderophore production and different iron concentrations in the medium [67,68].
Nitrogen is an essential and vital element for the normal growth and developments of plants. Many of the isolated PGPRs including species of Bacillus were found with the ability to fix atmospheric nitrogen. Syed-Ab-Rahman et al. [42] reported isolation of bacterial species including B. amyloliquefaciens (UQ154) and B. velezensis (UQ156) from soils of Arabidopsis plants. The Bacillus species were found with nitrogen-fixing ability. Other Bacillus species, which were isolated from Sophora Alopecuroides root nodules and rice showed nitrogen-fixing potential [69,70]. Likewise, in the present study, the isolated endophytic B. velezensis strain Lle-9 showed nitrogen fixation ability by growth on nitrogen-free medium. In addition, the strain was able to solubilize inorganic phosphate on a solid NBRIP medium supplemented with Ca 3 (PO 4 ) 2 as the sole source of phosphate. Phosphate solubilization is one of the most important plant growth-promoting traits associated with endophytic bacteria.
The different strains belonging to the genus Bacillus have been proved very effective as biocontrol agents and biofertilizers in sustainable agriculture [38,71]. Utilization of these bacteria has been found effective in controlling pathogenic microbes, exerting beneficial effects on plant growth and facilitating nutrient accessibility and assimilation [72][73][74]. Having these beneficial effects, the isolated strain Lle-9 was assessed for growthpromoting effects on the Asiatic lilium hybrid 'Tresor' under greenhouse conditions. Inoculated plants exhibited growth improvement and significant increases were observed for several growth parameters between the inoculated and non-inoculated control plants. Inoculated plants not only showed improved plant growth and bulb production but also produced a high number of flowers per plant. The improved growth performance in the Asiatic lilium hybrids 'Tresor' upon inoculation of Lle-9 might be due to the growth-promoting effects as were evaluated through several qualitative and quantitative tests. Previous studies also reported similar growth improvement when plants were inoculated with the isolated strains of Bacillus velezensis [75,40]. The isolated B. velezensis strain Lle-9 showed antagonistic effects against the broad-spectrum fungal pathogens. These enhanced antifungal activities might be due to the presence of a number of bioactive compounds, which have previously been recognized as antimicrobial in nature. Moreover, the strain Lle-9 exhibited several plant growth-promoting traits, which were reflected in the improved vegetative and reproductive growth of lily plants upon inoculation. Owing to these beneficial antifungal and plant growth-promoting properties, the B. velezensis strain Lle-9 may be a good choice to be utilized as a source of bio-fertilizer and bio-control agent in sustainable agriculture.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Corpus Luteum Color Doppler Ultrasound and Pregnancy Outcome in Buffalo during the Transitional Period

Simple Summary The advent of color Doppler ultrasonography promoted tremendous advances in research and clinical practice in animal reproduction, because it allowed noninvasive visualization of the vascularization in reproductive organs. In particular, the corpus luteum blood flow reflects luteal function better than luteal size in ruminants. Since buffaloes are a seasonal species, it is important to evaluate corpus luteum functionality also during the nonbreeding season, through blood flow examination and early pregnancy diagnosis. For this reason, we selected 29 Mediterranean buffaloes that had undergone synchronization and artificial insemination and were examined daily, from 5 to 10 days post-artificial insemination. Then, we retrospectively classified the buffaloes as pregnant or nonpregnant. Statistical analysis showed that pregnant animals had higher mean progesterone concentration and higher mean time average medium velocity values from Day 5 to Day 10 compared to nonpregnant buffaloes. Moreover, these two parameters could be used to predict the likelihood of pregnancy, starting on Day 6, although more reliable results could be obtained at Day 10 post-artificial insemination. In conclusion, a proper CL growth and development from Day 5 to Day 10 post-timed artificial insemination (TAI) is crucial for pregnancy maintenance during the transitional period. Abstract This study evaluated corpus luteum (CL) development in buffaloes out of breeding season and assessed an early pregnancy diagnosis. Mediterranean buffaloes (n = 29) were synchronized and artificially inseminated. CL B-mode/color Doppler ultrasonography examinations were performed daily from Days 5 to 10 post-synchronization, recording CL dimensions and blood flow parameters. Blood samples were collected on the same days for the progesterone (P4) assay. Data were grouped into pregnant or nonpregnant and retrospectively analyzed. The total pregnancy rate was 50.0% (13/26) on Day 45. A significant difference between CL average area in pregnant and nonpregnant buffaloes was recorded only on Day 10. Pregnant buffaloes showed a significantly higher mean P4 concentration and higher mean time average medium velocity (TAMV) values from Day 5 to Day 10 compared to nonpregnant buffaloes. Linear regression analysis showed a significant relationship between P4 levels and TAMV. Multiple logistic regression highlighted a significant influence of TAMV on pregnancy outcome, particularly on Day 8. This is probably due to the strong relationship between TAMV and P4 production. Both TAMV and P4 could be used to predict pregnancy starting on Day 6, although a more reliable result was obtained at Day 10. Thus, the period between Days 5 and 10 is critical for CL development during the transitional period in buffalo.

Introduction
The growing demand for meat and milk worldwide has caused a steady increase in the buffalo population in the last few years in both developed [1] and developing countries [2,3]. Buffaloes show high adaptability to different climates and conditions, and can be considered to be an important source of animal-derived protein for human consumption [1]. Although breeding is often managed on small farms, representing the basal rural economy in Asia, intensive breeding systems are performed in other countries such as Italy [4]. However, its productive efficiency is strongly related to the reproductive pattern, which is influenced by the photoperiod. Indeed, this species is considered to be a short-day breeder, meaning that it increases its reproductive activity when daylight hours decrease [4]. In Italy, these reproductive characteristics are opposite of the market requirement for milk production, suggesting that the application of the out-of-breeding season mating technique is responsible for a decline in reproductive efficiency, particularly during some periods of the year [4]. Embryonic mortality represents one of the main factors that are involved in pregnancy failure in both buffalo [5,6] and bovine [7] species. Several studies demonstrated that early development of the corpus luteum (CL) together with high progesterone (P 4 ) levels are involved in adequate embryonic growth and reduction of embryonic mortality [8,9]. In particular, a study performed during the breeding season showed that successfully pregnant buffaloes had larger CL development, higher P 4 concentrations, and increased blood flow to the CL, measured as the time average medium velocity (TAMV), compared to nonpregnant counterparts [10]. The latter experiment was performed during the breeding season from Day 5 to Day 10 post-timed artificial insemination (TAI). Furthermore, a low pregnancy rate was associated with delayed vascularization of the CL after Day 5. However, it cannot be ruled out that CL development occurs differently during the transitional period from mid-winter to spring when the females show a decrease in reproductive activity in response to increasing day length. The incidence of embryonic mortality during this period increases to 40% on some farms [11,12]. Therefore, the aims of this study were to: (i) evaluate CL development during the transition to the seasonal nadir in reproductive activity (transitional period from decreasing to increasing daylight length) through blood flow examination; (ii) determine whether CL blood flow parameters or progesterone levels can be used to estimate the likelihood of pregnancy by using the receiver operating characteristics (ROC) analysis, even if very early during CL development.

Animals
The Ethical Animal Care and Use Committee of the Federico II University of Naples approved the experimental design. The trial was performed in February 2018 on 29 pluriparous Italian Mediterranean buffaloes that were 120.2 ± 18.3 days in milk and bred on a commercial farm in the South of Italy between 40.5 • N and 41.5 • N parallel. The animals were maintained in open yards that allowed 15 m 2 per animal and 80 cm manger space. The buffaloes were fed a total mixed ration consisting of 55% forage and 45% concentrate, containing 0.91 milk forage units/kg of dry matter and 15% crude protein.
To assess the cyclic status, all the animals underwent two ultrasound examinations, spaced 12 days apart (MyLab 30Gold, Esaote, Genova, Italy) using a 7.5 MHz transrectal linear probe, before the start of the synchronization protocol. Only animals with a functional CL in at least one examination were included in the study.

Synchronization of Ovulation and Timed Artificial Insemination
Buffaloes were synchronized using the Ovsynch TAI program, which was developed for cattle [13] and is widely used in buffalo [14]. Briefly, 20 µg of gonadotropin-releasing hormone (GnRH) analogue (Buserelin acetate, 12 µg; Receptal ® , Intervet, Milan, Italy) was administered on Day 0 followed by a synthetic analogue of prostaglandin F2α (Cloprostenol ® , 0.250 mg/mL MSD, Milan, Italy) on Day 7 and an additional GnRH analogue administration on Day 9, with TAI on Day 10. All the animals underwent ultrasound examination on Day 10 of the synchronization protocol. Only those with a follicle that was larger than 1 cm and with a tonic uterus, with or without mucus vaginal discharge, were inseminated by the same experienced operator 20 h after the second GnRH injection. The dimensions of the preovulatory follicle were recorded on the day of TAI, and the follicle area was calculated according to the following equation: FL area = (a/2) × (b/2) × π (1) where: FL = follicle a = major axis of the follicle b = minor axis of the follicle Furthermore, ultrasound examinations were made one day post-AI to be sure that all the animals underwent ovulation.

Corpus Luteum and Blood Flow Evaluation
CL ultrasonography examinations were performed daily from Day 5 to Day 10 post-TAI using a portable ultrasound unit (MyLab 30Gold, Esaote, Genova, Italy) that was equipped with a 7.5 MHz linear transducer, designed for transrectal examination in large animals (LV 513). To obtain a better definition of the CL, once the ovary was visualized, the image was adjusted and then frozen to measure both the long and short axes. The CL area was calculated as follows: CL area = (a/2) × (b/2) × π, where a is the long axis; b is the short axis. To assess blood flow characteristics, the color Doppler mode was activated in order to evaluate the corpus luteum blood flow and, when the CL was clearly visualized, the sample gate was placed at the level of the primary branch of the ramus tubarius of the ovarian artery, just before hugging the corpus luteum. At this level, the artery was reasonably accessible and sufficiently large (2 to 3 mm in diameter) in order to measure flow rates accurately and in particular the flow indexes (TAMV, Resistivity Index (PI) and Pulsatility Index (PI); Figure 1). Color gain setting, velocity setting, and a color-flow filter setting were standardized during all the procedures and all the analyses were carried out by the same technician. In particular, the pulse repetition frequency was set at 0.7 kHz, the lowest possible that did not result in aliasing artifact, the power Doppler frequency was set to 1.5 kHz with a gain of 47, and the pulsed wave Doppler was set at 8 kHz, gain 67. Real-time B-mode/Color Doppler images of the continuous scans of the CL were recorded and then analyzed retrospectively. B-mode and color-flow mode short video clips (7 s duration) were stored in the internal memory of the ultrasound machine. Later, as described by Siqueira et al. [15], the CL area and the area of the cavity (where present) were assessed using the machine's internal calipers. To justify fluid-filled cavities, CLS was assessed by subtracting the area of the cavity from the entire CL area.

Progesterone Assay
Blood P4 levels were assessed using a radioimmunoassay (RIA) according to [16] to determine CL functionality. All the analyses were performed in the Laboratory of Veterinary Physiology of the University of Udine. Blood samples were collected from the jugular vein into heparinized tubes on

Progesterone Assay
Blood P 4 levels were assessed using a radioimmunoassay (RIA) according to [16] to determine CL functionality. All the analyses were performed in the Laboratory of Veterinary Physiology of the University of Udine. Blood samples were collected from the jugular vein into heparinized tubes on the same days as ultrasound examination (from Day 5 to Day 10). The samples were centrifuged at 800× g for 15 min and the plasma was stored at −20 • C until it was required for the P 4 assay, which was performed at the same time for all samples. The minimum detectable amount of P 4 was 2.2 ± 0.07 pg and the intra-assay and inter-assay coefficients of variation were 6.4% and 12.1%, respectively. Values of 1.2 ng/mL were considered to indicate the presence of a functional CL, according to previous studies [10].

Pregnancy Diagnosis
Pregnancy diagnosis was performed on Day 27 post-TAI using the same ultrasound machine as described above. Pregnancy status was confirmed on Day 45 and 70 post-TAI. Animals that were pregnant on Day 27 but not pregnant on Day 45 were considered to have encountered late embryonic mortality (LEM). Similarly, those that were pregnant on Day 45 and not pregnant on Day 70 were considered to have undergone fetal mortality.

Statistical Analysis
A repeated measure ANOVA was performed to evaluate the differences between pregnant and nonpregnant animals for P 4 levels, CL area, TAMV, PI, and RI values that were recorded from Day 5 to Day 10 [17]. Because of the low number of animals, the latter analysis was confirmed by another repeated measure ANOVA using a nonparametric approach [18,19]. Chi-square analysis was performed to evaluate differences in the pregnancy rate between buffaloes that showed an early or delayed increase in P 4 . A linear regression model was applied to evaluate the effects of TAMV, RI, PI, and CL area on P 4 blood levels, either on each day or throughout the experimental period. Moreover, a multiple logistic regression assay was performed to calculate the odds ratios for pregnancy using the TAMV, RI, PI, and CL area on P 4 blood levels as dependent variables [17]. All data were expressed as the mean ± standard error of the mean (SEM). Additionally, receiver operating characteristics (ROC) analyses were performed, focusing on CL characteristics (CL area, RI, PI, TAMV, and P 4 concentrations) on Day 5 to Day 10 post-TAI to identify the optimal cutoff value for predicting pregnancy. The latter was determined from the data point that minimized the distance and reported the relative sensitivity and specificity percentages and area under the curve (AUC) value.

Pregnancy Rate
Total pregnancy rate on Day 27 was 57.7% (15/26), and it was reduced to 50.0% on Day 45 after TAI. This means that the incidence of embryonic mortality was 13.3% (2/15). For the subsequent analysis, buffaloes that underwent LEM were included in the nonpregnant group. No fetal mortality was recorded. No differences were recorded between follicular area in both groups (data not shown). Average CL area was similar from Day 5 to Day 10 in pregnant animals compared to their nonpregnant counterparts (Table 1), although a (p < 0.05) difference was recorded on Day 10. Pregnant buffaloes also showed higher (p < 0.01) P 4 concentration for the Day 5 to Day 10 period compared to nonpregnant buffaloes, although significant differences were recorded from Day 7 on. The P 4 levels that were higher than 1.2 ng/mL on Day 5 post-TAI were recorded in 42.3% of buffaloes (11/26) and 81.8% (9/11) of them were pregnant. In 69.2% (9/13) of the pregnant buffaloes, P 4 concentrations that were higher than 1.2 ng/mL were recorded, whereas 30.8% (4/13) showed lower P 4 concentrations on Day 5 post-TAI. No effect of the day on pregnancy and no interactions were found with the repeated measures ANOVA.

Corpus Luteum Characteristics
No differences were recorded for the RI (0.41 ± 0.0 vs. 0.41 ± 0.0, in pregnant and nonpregnant buffaloes, respectively) and the PI (0.56 ± 0.0 vs. 0.55 ± 0.0, in pregnant and nonpregnant buffaloes, respectively) values, either on single days or throughout the experimental period. However, the mean TAMV value from Day 5 to Day 10 was (p < 0.01) higher in pregnant compared to nonpregnant buffaloes (14.05 ± 0.45 vs. 10.39 ± 0.57 cm/s), and significant differences were present from Day 6 ( Figure 2).  The characteristics of the CL blood flow that were evaluated by the eco-color Doppler technique showed a delayed vascularization in six buffaloes (19.2%). Blood flow features (TAMV, RI, PI) on Day 5 post-TAI were not recorded in these animals. Among these animals, only one buffalo (17.0%) was diagnosed as pregnant, whereas a higher pregnancy rate (60.0%; 12/20; p < 0.05) was recorded in buffaloes that showed a competent blood flow on Day 5 post-TAI.

Regression Analysis
The multiple linear regression analysis, considering the average of all parameters between Day 5 and Day 10, showed a significant relationship (R 2 = 0.394; p < 0.05) between P4 levels and TAMV. The characteristics of the CL blood flow that were evaluated by the eco-color Doppler technique showed a delayed vascularization in six buffaloes (19.2%). Blood flow features (TAMV, RI, PI) on Day 5 post-TAI were not recorded in these animals. Among these animals, only one buffalo (17.0%) was diagnosed as pregnant, whereas a higher pregnancy rate (60.0%; 12/20; p < 0.05) was recorded in buffaloes that showed a competent blood flow on Day 5 post-TAI.

Regression Analysis
The multiple linear regression analysis, considering the average of all parameters between Day 5 and Day 10, showed a significant relationship (R 2 = 0.394; p < 0.05) between P 4 levels and TAMV. The CL area tended (p = 0.096) to influence P 4 levels, according to the following equation: The same analysis was performed each day. In this case, the influence of TAMV and the CL area on P 4 levels was recorded only on Day 8 (R 2 = 0.303; p <0.05), according to the following equation: The multiple regression analysis in pregnancy that was performed using the average values from the period Day 5 to Day 10 of P 4 , CL area, TAMV, RI, and PI showed that pregnancy outcome was significantly influenced only by TAMV (odds ratio = 3.808; p < 0.01). If the multiple regression analysis was performed considering the same parameters on each day, a significant influence of TAMV was recorded also on Day 6 (odds ratio = 2.581), Day 7 (odds ratio = 1.899), and especially on Day 8 (odds ratio = 10.325).

Predicting Pregnancy
The ROC analyses indicated that both TAMV and P 4 were useful to predict pregnancy compared to other endpoints starting on Day 6 (Tables 2 and 3). Based on the distance from the ideal point, our analysis defined the best TAMV cutoff value as 11.88 cm/s for pregnancy prediction on Day 6 (sensitivity 76.9% and specificity 76.9%; p < 0.01). The P 4 cutoff value was 1.14 ng/mL for the pregnancy prediction on Day 6 (sensitivity 84.6% and specificity 61.5%; p < 0.05). However, Day 10 after TAI showed the best results for TAMV and P 4 in terms of sensitivity and specificity. The TAMV cutoff value was set at 13.19 cm/s, with an increase in sensitivity (92.3%) and specificity (84.6%; p < 0.01), while the P 4 cutoff value was set at 1.69 ng/mL, with an increase in sensitivity (92.3%) and specificity (92.3%; p < 0.01). For the other parameters, it was possible to set a cutoff value (2.38 cm 2 ) for the CL area only beginning on Day 10, with a sensitivity of 76.9% and a specificity of 53.8% (p < 0.05). It was not possible to set a cutoff value for RI and PI.

Discussion
This study analyzes the detailed CL development from day 5 to day 10 post-TAI in buffalo species during the transitional period to the seasonal nadir in reproductive activity. Although some studies of this type were performed [10], CL growth and characteristics were only investigated during the breeding season. Thus, we based our experimental design on that paper and we performed this study to confirm the results obtained by Neglia et al. [10] during the buffalo nonbreeding season. The CL is known to play a pivotal role in pregnancy establishment in several mammalian species, because of its capability to secrete P 4 [20]. The sensitivity to decreasing daylight hours deeply affects the reproductive efficiency of the buffalo, as demonstrated by the reduced CL functionality in some periods of the year [21,22]. This is responsible for the decline in reproductive efficiency and the increase in embryonic mortality [6].
The CL functionality can be assessed using several tools. The CL ecotexture that was assessed by ultrasound was one of the first approaches that was used to evaluate the CL [23,24], and the main limit of this technique was its subjective determination, which does not allow large-scale utilization. However, two reliable indicators of CL functionality are P 4 blood levels and/or the CL blood flow [25]. In our study, higher P 4 levels were recorded in pregnant buffaloes during the Day 7 to Day 10 period, which can be considered to be a critical window for embryonic growth and development. These results are in agreement with previous trials that were performed during the breeding season [10,25], in which the same trend was observed. High P 4 levels, and particularly an early increase in P 4 levels during the first days after mating, is responsible for larger embryonic development, high interferon-tau levels, and, consequently, reduced embryonic mortality, which represents one of the main causes of reproductive failure in buffalo [6,9,26]. This is also demonstrated by the evidence that animals that ovulated after treatment with a GnRH agonist on Day 5 after TAI showed an early increase in the P 4 concentration and a high pregnancy rate [11]. In our trial, significantly higher P 4 levels between pregnant and nonpregnant buffaloes were recorded starting from Day 7 and were definitely higher from Day 8. This interesting aspect further confirms the hypothesis that this period is critical for embryonic development, as previously observed [10]. Furthermore, more than 80% of buffaloes that showed P 4 levels that were higher than 1.2 ng/mL on Day 5 post-TAI were diagnosed as pregnant on Day 45, which strongly supports the evidence that an early increase in P 4 levels is fundamental for pregnancy outcome. It is still unclear if the CL dimensions may be considered to be a reliable indicator for P 4 production and/or the pregnancy rate.
Although CL area tended to be larger in pregnant buffaloes starting from Day 8 after TAI, a significant difference was only observed on Day 10 and similar values were recorded on average during the Day 5 to Day 10 period. The influence of CL dimensions on pregnancy rate is controversial. While in some studies, pregnant buffaloes showed a higher CL area compared to nonpregnant subjects [10,27], no differences were observed in other studies [28,29]. In a recent trial [30], a difference in CL dimensions between pregnant and nonpregnant buffaloes was observed only 14 days after insemination, while no differences were recorded during the Day 5 to Day 10 period. Therefore, further investigations are needed to clarify this aspect. Conversely, according to other authors, CL blood flow can be considered to be a consistent indicator of luteal functionality rather than its dimensions [31]. The results obtained in our trial further confirm this interesting aspect because TAMV values significantly affect the likelihood of pregnancy, as observed in previous studies [10]. After ovulation, the developing CL needs blood substrates for P 4 production and several studies support a strong correlation between blood flow and P 4 levels, either in cattle [32] or in buffalo [10]. However, a significant increase of blood flow has been correlated with pregnancy in cattle only 15 days after insemination [33], although some recent studies performed in Holstein cows highlight a difference between pregnant and nonpregnant cattle from Day 7 post-estrus [28]. In the present trial, TAMV values recorded on Day 8 post-TAI seem to significantly influence the likelihood of pregnancy in the buffalo species. This was supported by a recent study in which a significantly higher luteal blood flow and TAMV values were observed on Day 7 post-estrus in successfully pregnant Holstein cows, which were used as recipients for ET and treated with GnRH on Day 5, compared to nonpregnant counterparts [28]. However, according to these authors, the simultaneous evaluation of TAMV and the blood flow area may increase the accuracy of pregnancy prediction in cattle. In our trial, the blood flow area was not measured, but a significant influence of TAMV values was observed on Day 8, rather than Day 7. However, a delayed evaluation of blood flow on Day 8 cannot be ruled out, which may also increase the chances of pregnancy prediction in cattle. If these interesting findings are confirmed, this parameter may be used for an early pregnancy diagnosis either in cattle or in buffalo.
To determine the reliability of our results and to use them in a practical way, we used the ROC curve [34] to set a good cutoff value to predict pregnancy. Our results confirmed that both TAMV and P 4 blood levels at Day 6 could already provide some important information, but more reliable results in terms of sensitivity and specificity could be recorded directly on Day 10. Kanazawa et al. [29] made a similar experiment in cattle and reported that already at Day 7 it was possible to set up a cutoff value for both TAMV and P 4. Considering these findings, both TAMV and P 4 measurements seem to be better indexes of luteal function compared to the CL area. Moreover, the use of color Doppler ultrasonography could avoid stressful conditions for an animal that could not undergo blood sampling. We are aware that, very early during the CL development, we had cutoff values with low sensitivity and specificity. In our study, the highest values for sensibility and specificity among the days evaluated (5 to 10) were observed on Day 10. Actually, Day 10 may not be the best day to perform a nonpregnancy diagnosis based on CL vascularization, in the perspective of practical use. However, postponing this specific time window will delay the resynchronization of the open females and allow breeders to save money. Moreover, there will always be false positives, such as animals that, due to asynchronized ovulations, fail to respond to the TAI or that undergo embryo mortality. We are aware that further studies and a higher number of animals are necessary to confirm our results.

Conclusions
In conclusion, this study demonstrated that proper CL functionality and development from Day 5 to Day 10 post-TAI are crucial for pregnancy maintenance. The evaluation of CL blood flow using an eco-color Doppler technique is a noninvasive procedure that can be practically used to obtain useful information on CL functionality. Finally, the evaluation of the TAMV in the CL from Day 8 after TAI can be used to estimate likelihood of pregnancy, and thus to estimate the potential benefits of using an early resynchronization strategy to increase the number of pregnancies in buffaloes.


extfieldsofstudy: Biology Medicine

text:
A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs

African swine fever (ASF) is a devastating infectious disease in swine that is severely threatening the global pig industry. An efficacious vaccine is urgently required. Here, we used the Chinese ASFV HLJ/18 as a backbone and generated a series of gene-deleted viruses. The virulence, immunogenicity, safety, and protective efficacy evaluation in specific-pathogen-free pigs, commercial pigs, and pregnant sows indicated that one virus, namely HLJ/18-7GD, which has seven genes deleted, is fully attenuated in pigs, cannot convert to the virulent strain, and provides complete protection of pigs against lethal ASFV challenge. Our study shows that HLJ/-18-7GD is a safe and effective vaccine against ASFV, and as such is expected to play an important role in controlling the spread of ASFV. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s11427-020-1657-9 and is accessible for authorized users.

INTRODUCTION
African swine fever (ASF) is a devastating infectious disease in swine and has been catastrophic for the global pig industry. The causative agent, ASF virus (ASFV), belongs to the genus Asfivirus within the Asfarviridae family. ASFV has complicated architecture Alejo et al., 2018) and a large double-stranded DNA genome (170-193 kb) containing 151-167 genes depending on the strain. Depending on the B646L gene, which encodes the capsid protein p72, ASFs are divided into 24 different genotypes (Quembo et al., 2018), all of which have been detected in Africa (Quembo et al., 2018;Wade et al., 2019). Two of the ASFV genotypes have spread to other continents. The genotype I ASFV emerged in Europe in the 1950s and was eradicated from most European countries by the mid-1990s (Sánchez-Vizcaíno et al., 2013;Iglesias et al., 2017;Revilla et al., 2018). The genotype II ASFV was introduced into Georgia in 2007 and since then has become prevalent in many European countries (Iglesias et al., 2017;Pejsak et al., 2014); in 2018, the virus was transmitted to pigs in China and 10 other Asian countries (Wen et al., 2019;Ge et al., 2018;Le et al., 2019;Kim et al., 2020) (http://www.oie.int/). Pig production in China accounts for more than 50% of the pig population worldwide; however, over 99% of the pig farms in China are small-scale farms that produce less than 500 pigs annually, and these farms often have no biosecurity measures. Although the ASF outbreaks appeared to have been controlled by the slaughtering of infected pigs, the ASFVs widely contaminated the environment and, therefore, continued to pose a threat to the pig industry in China. Since no vaccine is available, the resumption of pig production is unlikely to succeed. Pig production in China in September 2019 was reduced by 40% compared with that in August 2018, and the price of pork has doubled since August 2019. The situation in other Asian countries may be the similar. These facts demonstrate that ASF cannot be controlled by culling infected pigs alone, and the development and application of an efficacious vaccine is urgently needed.

Protective efficacy of the attenuated ASFVs against virulent HLJ/18 challenge in pigs
To investigate whether the attenuated viruses could induce protective immunity, the HLJ/18-6GD-, HLJ/18-9GL&UKdel-, and HLJ/18-7GD-inoculated pigs were challenged intramuscularly (i.m.) with 200 PLD 50 of HLJ/18 virus; four 10-week-old SPF pigs were challenged similarly as a control. Body temperature and survival were recorded daily for 2 1 days. Blood, organs, and tissues including the heart, lung, spleen, tonsil, thymus and five lymph nodes (intestinal lymph node, inguinal lymph node, submaxillary lymph node, bronchial lymph node, and gastrohepatic lymph node) were collected from the dead pigs and from the surviving pigs, which were euthanized on day 21 post-challenge (p.c.) for viral DNA quantification by qPCR. The pigs in the control and HLJ/18-9GL&UK-del-inoculated groups all developed fever and died within 12 days p.c. (Figure 2A); high copies of viral DNA were detected in the blood, organs, and tissues from these pigs ( Figure 2B and C). Pigs in the HLJ/18-6GDand HLJ/18-7GD-inoculated groups all survived the duration of the observation period ( Figure 2A); viral DNA was detected in some organs or tissues of the pigs in these groups ( Figure 2D and E). It is noteworthy that the viral DNA copies in the HLJ/18-7GD-inoculated groups were lower than those in the other groups. One pig in each of the 10 3 TCID 50 and 10 5 TCID 50 HLJ/18-6GD-inoculated groups developed fever that lasted for 9 and 6 days, respectively ( Table 2). All four pigs in the 10 3 TCID 50 HLJ/18-7GD-inoculated group developed fever, but only one pig in the 10 5 TCID 50 HLJ/18-7GD-inoculated group had fever for 1 day (Table 2). These results indicate that HLJ/18-9GL&UK-del does not induce any protection, whereas HLJ/18-6GD and HLJ/18-7GD are immunogenic and able to protect pigs against lethal ASFV challenge. These data also indicate that the protection induced by HLJ/18-7GD is dose-related, whereas the protection induced by HLJ/18-6GD is not.

Safety evaluation of HLJ/18-6GD and HLJ/18-7GD in pigs
The biggest concern with a live attenuated vaccine is whether the vaccine seed virus could convert to a virulent strain during replication in vaccinated animals, since two early live vaccines, which were developed by attenuating virulent strain through serial passages in cell culture or from a naturally isolated avirulent strain, converted to lethal strains and caused 10%-50% mortality during their application in farmed pigs in Portugal and Spain in the 1960's (Coggins et al., 1968;Ribeiro et al., 1963). According to the guidelines for live attenuated vaccine development in China (http:// www.moa.gov.cn), a vaccine strain must be serially passed in the target animals for five passages to assess whether the animal-adapted seed strain will replicate more efficiently or become more lethal than the original seed strain. To this end, groups of six SPF pigs were intramuscularly inoculated with 10 7 TCID 50 of HLJ/18-6GD and HLJ/18-7GD viruses and observed for 21 days. Blood samples were collected from the pigs on days 5, 10, and 15 p.i. and spleen and lymph nodes were collected from each pig euthanized on day 21 p.i. to detect viral DNA by qPCR. In the HLJ/18-6GD-inoculated group, viral DNA was detected in the blood of five pigs and in the spleen of one pig (Table 3). However, in the HLJ/18-7GD-inoculated group, viral DNA was not detected in any samples collected (Table 3). All pigs survived the duration of the 3-week observation period. We next passed the HLJ/18-6GD-positive blood four more passages in pigs. Blood samples were again collected on days 5, 10, and 15 p.i., and organs and tissues including the heart, liver, spleen, lung, kidney, tonsil, thymus and lymph nodes from each pig euthanized on day 21 p.i. were collected for the detection of viral DNA. We found that the HLJ/18-6GD virus progressively replicated more efficiently in pigs, as evidenced by the increased number of viral DNA copies in the blood and the tissue distributions in pigs inoculated with passage 5 virus ( Figure 3A-D, Table 3). Of note, one pig in the passage 5 group for the HLJ/18-6GD virus developed severe disease and died on day 11 p.i. ( Figure 3F, Table 3). The blood that was harvested on day 5 p.i. from this pig was inoculated into five more pigs, and all of these pigs (passage 6) had high viral DNA copies in their blood and organs, four of them developed fever, and one of them showed severe disease symptoms and died on day 13 p.i. ( Figure 3E and F, Table 3). These results indicate that the HLJ/18-6GD virus has the potential to become more virulent during its replication in pigs.
We also blindly passed the viral DNA-negative blood of the HLJ/18-7GD-inoculated pigs for four more passages, but viral DNA was not detected in any samples collected from these pigs (Table 3). To further investigate the replication of HLJ/18-7GD in pigs, 14 seven-week-old SPF pigs were inoculated with a higher dose of 10 7.7 TCID 50 of the virus, and two pigs were euthanized on days 2, 5, 8, 10, 12, 16, and 21 p.i., respectively. The blood and organs of these pigs were collected for viral DNA detection. As shown in Figure 3, viral DNA was not detected in the blood, heart, liver, spleen, lung, kidney, tonsil, or thymus of any pig, but was detected in some lymph nodes of one or two pigs that were euthanized   Figure 3G). The lymph nodes that had the highest viral DNA copies were homogenized and inoculated into four more pigs, and two pigs were euthanized on day 8 p.i. and the other two were euthanized on day 10 p.i.. The blood, organs, and lymph nodes of these pigs were collected for viral DNA detection. As shown in Figure 3H, viral DNA was detected in two lymph nodes of one pig that was euthanized on day 10 p.i., but was not detected in any samples collected from the other three pigs ( Figure 3H). The virus was not detected in any subsequent pigs inoculated with the viral DNA-positive lymph node homogenates (data not shown). These results indicate that, after intramuscular injection, the HLJ/18-7GD virus is only maintained for a short period in certain lymph nodes of pigs, and does not appear in the blood or any other organs of pigs; therefore, HLJ/18-7GD is highly unlikely to convert to a virulent strain during its replication in pigs. Based on these safety evaluations, we selected the HLJ/18-7GD virus as a vaccine strain for ASF and performed additional studies in clean commercial pigs.

Protective efficacy of HLJ/18-7GD against virulent HLJ/18 challenge in commercial pigs
Since the protective immunity induced by a single dose of vaccine may decline in a few weeks, a second dose may be needed in actual animal disease control practice. In addition to intramuscular challenge, we also evaluated the protective effects of challenging pigs orally, as the oral route is how pigs are mainly infected with ASFV in nature. Groups of 7week-old piglets from a local farm were transferred to a P2 facility and vaccinated once or twice (with a 3-week interval) with 10 5 TCID 50 of the vaccine and were then moved to a P4 facility for challenge at different timepoints after vaccination. Groups of four or five similar aged unvaccinated pigs were used as controls. All pigs were observed for three weeks after challenge with the lethal HLJ/18 virus. Samples, including the blood, heart, spleen, lung, tonsil, thymus, and lymph nodes, from dead pigs and surviving pigs that were euthanized on day 21 p.c. were collected for viral DNA detection by qPCR. Of note, the control pigs in all of the challenge studies died within 11 days of challenge, and high viral DNA copy numbers were detected in all of the organs tested from these pigs ( Figure 4, Table 4). As shown in Figure 4A, the pigs that were inoculated with one dose of 10 5 TCID 50 vaccine and challenged i.m. with 200 PLD 50 of HLJ/18 on day 28 post-vaccination all survived the 3-week observation period, but viral DNA was detected in the blood, tonsil, and two lymph nodes of one of the five pigs that were euthanized at the end of the observation period ( Figure 4A), indicating that HLJ/18-7GD provides similar protection in both farmed and SPF pigs. Two groups of pigs were inoculated twice with 10 5 TCID 50 of the vaccine: one group was challenged i.m. with 200 PLD 50 of HLJ/18 two weeks after the second dose, and the other group was challenged orally with 10 6.5 HAD 50 of HLJ/ 18 three weeks after the second immunization. All vaccinated pigs survived the 3-week observation period, and very low levels of viral DNA were detected in one lymph node of one pig in the i.m. challenge group ( Figure 4B); viral DNA was not detected in any pigs in the orally challenged group ( Figure 4C). These results indicate that two immunization with 10 5 TCID 50 HLJ/18-7GD could provide solid protection against lethal HLJ/18 challenge in pigs. Commercial pigs are usually raised for at least five months. When we challenged a group of pigs that were vaccinated twice with 10 5 TCID 50 HLJ/18-7GD at the age of five months (80 days after the last vaccination), we found that 80% of them survived the challenge, but long-lasting immunity was unable to efficiently clear the challenging virus ( Figure S1 in Supporting Information). The data in Figure 2 show that the protection induced by HLJ/18-7GD was dose-related, and therefore, we investigated whether a higher inoculation dose could induce better long-term protection. To this end, we inoculated a group of six pigs with one 10 6 TCID 50 dose of the vaccine and challenged the pigs at 10 weeks post-vaccination. We found that all of the vaccinated pigs remained healthy and survived the duration of the observation period, and low levels of viral DNA were detected in one lymph node of two different pigs, but not in the other four pigs ( Figure 4D). These results indicate that the long-lasting immunity induced by a single dose of 10 6 TCID 50 of HLJ/18-7GD could provide solid protection against lethal ASFV challenge, and suggest that two doses of 10 6 TCID 50 of HLJ/18-7GD administrated appropriately could protect pigs for their entire lives.

Safety evaluation of HLJ/18-7GD in pregnant sows
In practice, to control ASF, sows will also need to be vaccinated. There is always the potential for vaccine inoculation to cause problems in pregnancy. We therefore investigated the safety of the vaccine in pregnant sows. Six primiparous sows at different pregnant stages were inoculated with 10 6 TCID 50 of HLJ/18-7GD: one sow was inoculated on the 35th day of pregnancy (early gestation), two sows were inoculated on the 94th day of pregnancy (late gestation), and three sows were inoculated on the 63rd day of pregnancy (mid-gestation). Two sows were left untreated as control (Table 5). The pregnant sows were monitored daily from the day of inoculation to four weeks after the piglets were delivered. All sows remained healthy after inoculation and delivered their piglets on the expected dates (Table 5), indicating that inoculation with HLJ/18-7GD will not cause disease or abortion in pregnant sows or affect the health of the piglets.
Similar to the findings with ASFV Georgia/2007 and Benin 97/1 (Reis et al., 2016;O'Donnell et al., 2015a), our HLJ/ 18-6GD in which the MGF360 and MGF505 genes are deleted was also attenuated in pigs and protected pigs against challenge with the virulent parental virus; however, our safety evaluation data indicated that HLJ/18-6GD has a high risk converting to a virulent strain, and therefore, should not be used as a vaccine. Fortunately, for the HLJ/18-7GD virus, this risk has been eliminated by deletion of the CD2v gene.
Previous studies indicated that ASF vaccines easily lost their immunogenicity after adaptation in cell lines and could not confer protection against homologous virus challenge in pigs Gallardo et al., 2018;Krug et al., 2015), suggesting that the ASF vaccine should be produced in primary cells, which may limit large-scale production. The porcine bone marrow (PBM) cells have been reported to support ASFV replication (Malmquist and Hay, 1960). We found that PBMs supported the growth of HLJ/18-7GD well and at least 200,000 doses of vaccine (10 6 TCID 50 /dose) could be produced from PBMs derived from one SPF piglet. Moreover, we found that the HLJ/18-7GD maintains its immunogenicity after being serially passed in PBMs for six passages (data not shown). Therefore, using the primary Figure 3 Safety evaluation of HLJ/18-6GD and HLJ/18-7GD in pigs. HLJ/18-6GD and HLJ/18-7GD were serially passed in pigs, and the indicated samples from the dead pigs or surviving pigs that were euthanized on day 21 post-inoculation (p.i.) were collected for viral DNA detection. The viral HLJ/18-6GD DNA copies from the 2nd to 6th passage are shown in panels A to E, respectively. The survival rate of pigs inoculated with different passages of HLJ/ 18-6GD is shown in panel F. Viral DNA was not detected in any samples collected from the HLJ/18-7GD initially inoculated pigs or the blindly passed pigs (Table 3). Therefore, 14 pigs were inoculated with the 10 7.7 TCID 50 dose of HLJ/18-7GD and two pigs at each timepoint were euthanized on days 2, 5, 8, 10, 12, 16, and 21 p.i.. Viral DNA was detected in a few lymph nodes of some pigs (G), and in two lymph nodes of one pig after the second passage (H), but was not detected in the blood, heart, liver, spleen, lung, kidney, tonsil, thymus, or other lymph nodes of any pigs (data not shown). The dashed lines indicate the lower limit of detection. LN 1: intestinal lymph node; LN 2: inguinal lymph node; LN 3: submaxillary lymph node; LN 4: bronchial lymph node; LN 5: gastrohepatic lymph node; LN 6: mediastinal lymph node. PBMs for large-scale production of HLJ/18-7GD is feasible and cost-effective.
In summary, we used the Chinese ASFV HLJ/18 as a backbone to generate six viruses bearing different gene deletions, and found that HLJ/18-7GD, which has seven genes deleted, is fully attenuated in pigs, has a low risk of converting to a virulent strain, and could induce solid protection in pigs against lethal ASFV challenge. HLJ/-18-7GD has been fully evaluated and proven to be safe and effective against ASFV. We therefore expect that this vaccine will play an important role in the control of ASFV.

Ethics statements
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. The protocols were approved by the Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS) and the Animal Ethics Committee of Heilongjiang Province, China.

Biosafety statement and facility
All experiments with live ASF viruses were conducted within the enhanced biosafety level 3 (P3+) and level 4 (P4) facilities in the HVRI of the CAAS approved by the Ministry of Agriculture and Rural Affairs and China National Accreditation Service for Conformity Assessment.

Cell culture, viruses, and pigs
Primary porcine alveolar macrophages (PAMs) were collected from 30-40-day-old specific-pathogen-free (SPF) pigs, and the cells were maintained in 10% FBS RPMI 1640 medium (Thermo Scientific, USA) at 37°C with 5% CO 2 . Peripheral blood mononuclear cells (PBMCs) were prepared from EDTA-treated swine blood by using a pig PBMC isolation kit (TBD Sciences, China). Porcine bone marrow (PBM) cells were collected as described previously   (Malmquist and Hay, 1960). ASFV Pig/Heilongjiang/2018 (HLJ/18) was isolated from field samples in China as described previously . The HLJ/18 stock used for challenging studies was prepared from the defibrinated blood of virus-inoculated SPF pigs and titrated in PAMs. SPF Large White and Landrace-crossed pigs were obtained from the Laboratory Animal Center of the HVRI. Clean commercial pigs and pregnant sows were obtained from local farms where the animals were tested to ensure they were negative for porcine respiratory and reproductive syndrome (PRRS), classical swine fever (CSF), ASFV, and pseudorabies virus (PRV).

Generation of gene-deleted ASFVs
Gene-deleted ASFVs were generated by homologous recombination between the parental ASFV HLJ/18 genome and recombination transfer vectors by transfection and infection procedures. Recombinant transfer vectors containing the flanking genomic regions of the targeted genes, mapping approximately 500-1,000 bp to the left and right of these deleted parts, and the reporter gene cassettes containing the reporter genes of eGFP or mCherry with the ASFV p72 late gene promoter or CD2v gene promoter were used as shown in Figure 1. Recombinant transfer vectors were constructed by using fusing PCR and the Gibson Assembly technique (Invitrogen Life Sciences, USA). PAMs were transfected with the constructed transfer vectors by using the TransIT-LT1 transfection reagent (Mirus Bio, Madison, WI, USA) and then infected with HLJ/18 at 24 h post-transfection as described previously with minor modifications (Reis et al., 2017;O'Donnell et al., 2015a;Monteagudo et al., 2017;O'Donnell et al., 2015b). These constructions created the expected deletions in the viral genome as shown in Figure 1. HLJ/18-9GL&UK-del, HLJ/18-CD2v&UK-del, and HLJ/ 18-7GD with two deleted parts of the viral genome were constructed by using two successive homologous-recombination procedures. The resulting virus from each homologous recombination event was purified by successively picking fluorescent plaques combined with limited dilution on monolayers of PAMs. The virus obtained from the last round of purification was amplified in PAMs to make a virus stock. To ensure the absence of parental HLJ/18 and the desired deletions in each recombinant genome, virus DNA was extracted from the virus stock and confirmed by PCR and sequencing.

Virus titration
The wild-type HLJ/18 virus was quantified by using the hemadsorption (HAD) assay as described previously (Malmquist and Hay, 1960) with minor modifications. PBMCs were seeded in 96-well plates. The samples were then added to the plates and titrated in triplicate using 10-fold serial dilutions. HAD was determined on day 7 post-inoculation (p.i.), and 50% HAD doses (HAD 50 ) were calculated by using the method of Reed and Muench (Reed and Muench, 1938).
Samples from the gene-deleted ASFV-infected pigs or cell supernatants were quantified by testing their 50% tissue culture infectious dose (TCID 50 ). PBMCs were seeded into 96-well plates, and three days later 10-fold serially diluted samples were added into each well in triplicate. After seven days of culture, the fluorescent protein expression was assessed by using fluorescence microscopy (Axio Observer.Z1, Zeiss, Germany). TCID 50 was calculated by using the method of Reed and Muench. qPCR ASFV genomic DNA was extracted from cell supernatants, tissue homogenate, or EDTA-treated whole peripheral blood by using GenElute™ Mammalian Genomic DNA Miniprep Kits (Sigma Aldrich, USA). qPCR was carried out on a QuantStudio 5 system (Applied Biosystems, USA) according to the OIE-recommended procedure.

Pig studies
To determine the 50% pig lethal dose (PLD 50 ) of HLJ/18, five groups of four 50-day-old SPF pigs were inoculated intramuscularly (i.m.) with 10-fold serial dilutions containing 10 0 to 10 4 HAD 50 of virus in a 1 mL volume. The pigs were monitored daily for 21 days for temperature and mortality. The PLD 50 was calculated by using the method of Reed and Muench.
To evaluate the virulence of the gene-deleted ASFVs in pigs, groups of 50-day-old SPF pigs were i.m. inoculated with 10 3 TCID 50 and 10 5 TCID 50 of each test virus, and the pigs were monitored daily for 21 days for temperature and mortality.
To evaluate the protective efficacy of the gene-deleted virus against virulent ASFV challenge, groups of pigs were i. m. inoculated once or twice with the indicated dose of genedeleted ASFV, and were then challenged with lethal HLJ/18 virus at the indicated timepoint. Similar aged untreated pigs were challenged as controls. The pigs were monitored daily for 21 days post-challenge (p.c.) for clinical signs and mortality. Blood, organs, and tissues including the heart, liver, spleen, lung, kidney, tonsil, thymus, and lymph nodes from the dead pigs or surviving pigs that were euthanized at the end of the observation period were collected for the detection of viral DNA by using qPCR as recommended by the OIE (King et al., 2003).

Safety evaluation of the gene-deleted ASFVs as live-attenuated vaccines in pigs
HLJ/18-6GD and HLJ/18-7GD were evaluated for safety in SPF piglets by following the recommendations of the OIE described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2019 (https://www.oie.int/standard-setting/terrestrial-manual/access-online/) and the guidelines for live attenuated vaccine development in China (http://www. moa.gov.cn/nybgb/2006/dbq/201806/t20180616_6152332. htm) with minor modifications. Briefly, groups of six pigs were intramuscularly inoculated with 10 7 TCID 50 of test virus. Blood samples collected from pigs on days 5, 10, and 15 p.i. and organs and tissues collected from pigs that were euthanized on day 21 p.i. were used for viral DNA detection. Viral DNA-positive blood samples collected from the HLJ/ 18-6GD-inoculated pigs were used to inoculate pigs for the subsequent passage. Since viral DNA was not detected from any pigs that were inoculated with the HLJ/18-7GD virus, the pooled blood samples collected from pigs on days 5 and 10 p.i. were used to inoculate pigs for the subsequent passage.
To further evaluate the safety of HLJ/18-7GD in pigs, 14 50-day-old SPF pigs were i.m. inoculated with the 10 7.7 TCID 50 of HLJ/18-7GD, and then two pigs were euthanized on days 2, 5, 8, 10, 12, 16, and 21 p.i., respectively, and their samples, including the blood, heart, liver, spleen, kidney, tonsil, thymus, and six lymph nodes (intestinal lymph node, inguinal lymph node, bronchial lymph node, submaxillary lymph node, gastrohepatic lymph node, and mediastinal lymph node) were collected for viral DNA detection by qPCR. The viral-positive organs or samples were collected, homogenized, and used to inoculate subsequent pigs. The pigs after the third passage were viral DNA-negative; their lymph nodes were pooled, homogenized, and used to inoculate four SPF pigs for one more passage (the fourth passage). The pigs were monitored daily for temperature and clinical signs for 21 days, and then were euthanized to collect blood and the indicated tissues and lymph nodes for virus detection.

The safety of HLJ/18-7GD in pregnant sows
To determine the safety of HLJ/18-7GD in pregnant sows, eight primiparous sows at different stages of gestation were purchased from a local pig farm. One sow of early gestation (the 35th day of pregnancy), two sows of late gestation (the 94th day of pregnancy), and three sows of mid-gestation (the 63rd day of pregnancy) were i.m. inoculated with 10 6 TCID 50 of HLJ/18-7GD. Two sows of early gestation were untreated as controls. All sows were monitored daily for temperature and clinical signs until four weeks after the piglets were delivered.

Compliance and ethics
The author(s) declare that they have no conflict of interest.


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Study on horizontal diffusion of agent solutions in underground unsaturated soil: experiments and model simulations

The solute transport or water transport in unsaturated soil plays an important theoretical guiding role in remediation engineering. In order to study the diffusion of agent solutions in practical remediation engineering, the soil column experiments of unsaturated soil were carried out using the self-made laboratory test device. The effects of injection rate and soil pressure (confining pressure) on the solute diffusion in soil were studied. The experimental results show that the injection velocity has a great influence on the solute diffusion in soil, and the change in the diffusion rate is not proportional to the injection velocity. The effect of confining pressure on solute diffusion was first promoted and then suppressed. The fractal Richards equation (FRE) model was used to fit the water content curves. The results show that the FRE model has a fine fitting effect on this series of experiments, proving that it can provide model support for the simulation of solute diffusion in underground unsaturated soil. At the same time, the physical significance reflected by the fitting parameters can be used to analyze the mechanism of the experimental phenomena.

pollutant transport. These studies show that confining pressure is one of the crucial factors 1 affecting the diffusion of pollutants or agent solutions in underground soil. Feng Chu [15] 2 investigated soil-water characteristics of unsaturated undisturbed loess under isotropic 3 compression condition, and influences of net confining pressure on matric suction characteristics 4 of unsaturated undisturbed loess are determined and illustrated. And matric suction on water 5 transport and solute transport in soil is also self-evident [16]. To sum up the previous studies, we 6 can note that the influence of confining pressure on soil consolidation and pore water pressure 7 indirectly affects the diffusion performance of water and solute in soil. Therefore, it is necessary 8 to explore the law of water transport or solute transport in underground soil under different 9 confining pressures, and the confining pressure of soil in laboratory experiments was achieved 10 by applying external forces. 11 Considering the complexity of the actual engineering environment and the urgency of [24] developed a simple method, a horizontal one-dimensional infiltration test, to estimate water diffusivity of unsaturated soil. Moreover, several models have been proposed for the study of 1 water transport [25,26]. The classical Richards model [27], proposed by Richards in 1937, is a 2 widely recognized mathematical model of water transport in unsaturated soil. Many researchers 3 have modified and simplified the Richards model, and various mathematical models have been 4 proposed [28, 29] based on the experimental results. The water or solute transport in unsaturated 5 soil is often influenced by the complex pore structure of soil and the external conditions. 6 Previous studies have shown that particle distribution, pore structure and pore connectivity in 7 soil all present fractal characteristics [30][31][32]. More and more researchers [33][34][35]  would not be weakened by the accumulation of time or space distance, and the water or solute 14 transport could be predicted accurately. Therefore, it is necessary to explore whether the FRE 15 model can be used to characterize the diffusion law of water or solute in unsaturated soil under 16 complex conditions, and whether its parameters can reflect physical properties. It is very 17 important to solve the problem of insufficient diffusion and to predict the transport of agents in 18 soil.

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In summary, the study on the water transport or solute transport in unsaturated soil is to 20 explore the diffusion law of agents in underground soil in actual remediation engineering.

21
Transport of water or solute in unsaturated soil plays an important theoretical guiding role in remediation engineering. Therefore, it is extremely important to carry out quantitative laboratory 1 simulation experiments and to find a suitable and effective mathematical model. In this paper, a 2 technical parameter (injection rate) and one physical property (confining pressure) of 3 contaminated soil are taken as research objects, and a series of experiments considering two 4 influencing factors are carried out. On this basis, the FRE model was used to characterize the 5 water retention curves, by fitting the experimental data. The rest of this work is organized as

12
Considering that the diffusion of agents in soil is symmetrical in all horizontal directions, this 13 paper chooses one-dimensional horizontal diffusion as the research object. A solution of sodium 14 phosphate (Na 3 PO 4 ) was injected, as the remediation agent. The soil water content was measured 15 in real time by using the soil water content detector in each test hole of equal distance. Data was 16 collected and recorded in the process of experiments.

19
The principle of high pressure jet grouting technology is to inject chemical agents into 20 underground soil by air flow and liquid flow with high pressure. And then pollutants are oxidized 21 by the chemical oxidant (the remediation agent), degraded or transformed to low toxic products.
The effective action range of high-pressure jet grouting technology includes core cutting part, 1 diffusion part or extrusion part. As shown in Fig. 1, the effective range is a cylindrical 2 consolidation body composed of the agent and soil mixing slurry body, the stirring mixing part 3 and the diffusion part [5]. However, it is understood that core cutting part will mainly depend on 4 the cutting action of the jet and on the soil resistance to disaggregation. This is mainly a physical 5 effect on soil, extremely complex to be quantified [38]. In practical projects, the radius of 6 effective range in the horizontal direction is often to measure the remediation effect and 7 efficiency. Therefore, the study on the water or solute transport in underground soil mainly 8 focuses on the diffusion part in the horizontal direction, and the diffusion in the vertical direction 9 is not considered in this paper. The diffusion conditions were simulated by laboratory 10 experiments, and influences (injection rate and confining pressure) were discussed.

13
The laboratory scale experiment of water transport in horizontal unsaturated soil column, the 14 most widely used in experimental research, can eliminate the influence of gravity on water 15 transport in unsaturated soil, which can make the influencing factors of water transport single 16 and parameters more accurate. Horizontal soil column method is a laboratory method for the 17 determination of unsteady flow in soil hydraulic diffusion, which was first proposed by Bruse

19
In order to minimize the error between laboratory experiments and actual situations, the 20 experimental soil used in this paper was undisturbed. Soil samples were shallow soil, taken from 21 the middle area of Jiangsu province. After removing impurities and drying, soil samples were reconfigured to meet the required water contents. The initial water content of undisturbed soil 1 was 17.58%. When the in-situ remediation is carried out by high-pressure jet grouting 2 technology, the agent is required to be water-soluble, relatively stable under high-pressure 3 conditions, and has little side effect on the environment [5]. Sodium phosphate (Na 3 PO 4 ) 4 selected in this paper is a kind of inorganic compound, and it is easily soluble in water (its 5 solubility is 28.3 g/100 mL). 6 In view of problems in practical engineering application of high-pressure jet grouting 7 technology, such as agent overflow and insufficient diffusion, which are mainly affected by 8 injection rate and confining pressure. Therefore, two factors are selected to study the influence 9 on the diffusion rate of agents in soil, namely, injection rate (60 ml/min, 40 ml/min, 20 ml/min), 10 and confining pressure (5 kPa, 10 kPa, 15 kPa, 20 kPa, 30 kPa, 40 kPa, 50 kPa).

11
The experimental pipe was placed vertically to fill with the prepared soil samples and where,  is the water content,   D  is the water diffusivity, which is assumed to only related to 16 water content, x is the transport distance of water in soil, and t is the ordinary time. The  Where the dimensionless exponent The transport of water in unsaturated soil is often affected by the complex pore structure 6 and boundary conditions inside the soil, and the pore structure is not fixed during the transport 7 process of water. So, it is difficult to describe the transport law of water in unsaturated soil where the initial condition is    Table S1. Fig. 4 indicates that FRE can describe the trend of water transport in soil column 1 experiments under three different injection rates, and they fit well at all positions. The R-square 2 and RMSE in Table S1 were computed by pooling data from all curves shown.

3
The parameter n of the FRE is positively correlated with the change rate of water content 4 in soil [37]. And, the parameter n in the three groups of experiments is drawn as the change 5 curves in Fig. 5. Fig. 5 shows that the lower injection rate is, the faster the water content of soil 6 changes, that is, the faster the diffusion rate of the simulated agent in the soil column increases, 7 which is consistent with the law reflected in Fig. 4. In addition, the R-square and RMSE in Table   8 S1, reflecting a fitting effect, show that the FRE fits the water transport under a low injection rate 9 more closely than the high group. Especially, in the experimental group (c) with higher injection 10 speed, it can be found that the fitting effect of the forward position (X = 200 mm, 400 mm…) is 11 significantly decreased.

12
The diffusion of solute or transport of water in soil mainly flows through pore channels 13 formed by the liquid phase of soil and self-infiltration [49]. With the increase in injection rates, 14 more and more pore channels will participate in the diffusion. When the injection rate is too high, 15 pore channels are not enough to pass through a large amount of liquid, so the growth rate of 16 diffusion will gradually slow down. Moreover, due to the injection of a large amount of liquid in 17 a short period of time, the liquid that has no time to diffuse and gradually accumulates, which 18 will cause a certain degree of damage to the originally stable structure of the soil, and the pore 19 channels used for liquid diffusion and transfer will also be damaged to a certain extent. When the 20 injection rate is relatively low, the agent has enough time to permeate and diffuse, and it provides 21 a stable environment for the agent filled in pores of the soil to react with pollutants. Therefore, with the decrease of injection rates, the remediation is improved and the utilization rate of agents 1 will be higher.

2
In actual construction processes, on the one hand, sampling should be carried out in advance 3 to determine the optimal injection rate. On the other hand, if the remediation agent is expensive, 4 the injection speed should be appropriately reduced on the premise of the remediation effect.

5
Balancing the time cost and economic cost is to achieve the maximum benefit. In engineering application, the high-pressure jet grouting technology is mostly used in the 10 polluted soil with a depth of 0 ~4 m (the corresponding confining pressure of soil is 0~ 40 kPa).

11
Confining pressure is an important factor affecting the diffusion of agents in underground soil.

12
Therefore, the influence of a certain confining pressure (no higher than 50 kPa) on solute 13 transport in subsurface soil will be considered in this paper.
14 In this group of experiments, the agent solutions with the concentration of 15 g/L was 15 injected at the rate of 10 ml/min, and the pressure diffusion was carried out in the soil sample 16 with the initial water content of 15%. Fig. 6 shows the time for the water content of soil samples 17 to reach 20% after agents diffusion, under various confining pressures. It can be seen from Fig. 6 18 that the time for the diffusion of agents decreases first and then increases with the increase of 19 confining pressure of soil, that is, the diffusion velocity of agents increases first and then 20 decreases with the increase of confining pressure. In addition, there is an obvious optimal 21 confining pressure value (15 kPa) in this group of experiments. By decomposing Fig.6 into Fig.  6(a) and Fig. 6(b), it can be clearly seen that when the confining pressure of soil is 15 kPa, the 1 diffusion time of remediation agent is the shortest. In the process of diffusion, the appropriate 2 increase of confining pressure can promote the diffusion of agents in underground soil. And 3 when the confining pressure increases gradually, the effect of promoting diffusion decreases 4 gradually, and it presents a restraining trend when the confining pressure is larger.  Table S2.
14 The fractal order α of the FRE can characterize the underlying water transport 15 environment property in heterogeneous soil, and CHEN Wen [47] pointed out that the smaller 16 the value of α, the more complex the water transport environment would be. Fig. 8 shows curves 17 of parameter α, fitted by FRE in each group, and according to the experimental law shown in Fig.   18 6, they are also divided into two groups: (a) and (b). Fig. 8 indicates that with the increase of the 19 confining pressure, values of α first decreases (Fig. 7(a)) and then increases (Fig. 7(b)), which is 20 consistent with Fig. 6. This represents that the rate of water transport is related to the complexity of soil environment caused by the confining pressure, which can also be seen from the change of 1 parameter C 0 in Table S2.

2
After the agent solution is injected into the soil column, it diffuses from the injection 3 point to all directions. For the reason of the horizontal loading pressure in this experiment, 4 proximal soil samples will be compressed first and pores will be reduced. Since liquid diffusion 5 is mainly formed by filling soil pores to form water channels, so compact pores of soil particles 6 shrinking make diffusion easier, and the diffusion speed tends to increase with the increase of 7 confining pressure. When the confining pressure reaches a certain value, the pores in all 8 directions are squeezed to a certain extent, and the compactness of soil reaches a certain degree.

9
The shrinkage of pores in the soil will lead to the obstruction of flow paths for the liquid, and the 10 diffusion of agents becomes very difficult. At this time, with the increase of the confining 11 pressure, the diffusion rate of agents will gradually slow down.

12
Due to different confining pressures on contaminated soil at different depths in 13 engineering projects, the diffusion in the remediation process is also different, leading to 14 different remediation effects. For the shallow underground soil, some confining pressure can 15 promote its diffusion. Owing to the deep underground soil under the higher confining pressure, 16 the diffusion velocity will be restrained to some extent. Therefore, through changing other 17 external conditions, the diffusion can be accelerated to achieve an optimal remediation effect.  That is to say, it exists a confining pressure value to optimize the diffusion velocity. Under the 7 high confining pressure, due to the compaction of soil, non-uniform diffusion and accumulation 8 of agents will occur, and the effect of remediation is highly uncertain.

9
In addition, water content curves were presented, fitting the FRE from the experimental