Prickly Ash Seed Kernel: A New Bio-Fumigation Material Against Tobacco Black Shank

The present study evaluated the fumigant effect and potential mechanisms of Chinese prickly ash seed kernel (PSK) against tobacco black shank caused by Phytophthora nicotianae Breda de Haan. The results showed that PSK fumigant increased the soil organic matter, hydrolysable nitrogen, available potassium, and total phosphorus significantly, while the pH was decreased. Application of 2% PSK promoted plant growth, with higher plant height, root length, and dry weight compared to those in the treatment with no PSK. Both in vitro and in vivo tests indicated that PSK fumigant effectively inhibited mycelial growth of P. nicotianae, and significantly reduced the severity of tobacco black shank. Based on the analysis of volatiles in PSK by headspace solid-phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS), 28 components were identified, among which monoterpenes were the major components (accounting for 79.46% of peak area). Among the six top components, linalool, piperitone, sabinene, and limonene exhibited strong mycelial inhibition of P. nicotianae. Linalool was the most effective with an IC50 value of 18.03 μL/L. Results of GC-MS revealed that the main components of PSK extract were cis-Vaccenic acid (40.81%), n-Hexadecanoic acid (15.67%), 9,12-Octadecadienoic acid (Z,Z)- (15.25%), Ethanone-1-(2-hydroxy-4,6-dimethoxyphenyl)- (10.32%), cis-9-Hexadecenoic acid (6.21%), and Psilocin (11.75%). Among them, Ethanone-1-(2-hydroxy-4,6-dimethoxyphenyl)- and 9,12-Octadecadienoic acid (Z,Z)- have an obvious inhibitory effect on P. nicotianae. Additionally, changes in soil bacterial and fungal communities were observed. The relative abundance of some microbes, such as Pseudomonas, Azospirillum, Thermomonas, Glutamicibacter, Dyella, and Trichoderma, increased, which have many beneficial microbes in these genera, indicating that microbial community shift could be one of the important outcomes of the PSK fumigant effect. In conclusion, PSK could be a new bio-fumigation material against plant soilborne pathogens, which can function as both a fungicide and fertilizer.


Introduction
Tobacco black shank is a critical soilborne oomycete disease caused by Phytophthora nicotianae Breda de Haan [1]. In China, the epidemic area of the disease is more than 76,372 ha, and the direct economic loss is above a billion dollars. Currently, the application of chemical fungicides, such as

HS-SPME GC-MS Method
The extract of PSK was prepared by ethanol-petroleum ether method, according to Dong et al. (2016) [13]. Headspace solid-phase microextraction (HS-SPME) was chosen as the method for volatile compound sampling. The so-called sandwich-type (Divinylbenzene/Carboxen/Polydimethylsiloxane, Supelco, Bellefonte, PA, USA) fiber was used for the absorption of PSK volatile compounds. For sampling volatile compounds, the SPME fiber was inserted into the headspace of sample vial containing 10 g PSK at 25 • C for 20 min. When the sampling was completed, the SPME fiber was then removed from the sample vial and immediately inserted into the gas chromatography (GC) injector at 250 • C for 5 min with a split ratio of 10:1. The used SPME fiber was conditioned at 250 • C for 5 min prior to the next sampling. The gas chromatography-mass spectrometry (GC-MS) analyses were done with an Agilent 7890-5975C GC-MSD system (Santa Clara, CA, USA) with a DB-5 MS fused-silica capillary column (30 m × 0.25 mm i. d, 0.25 µm film thickness). The injection volume was 1.0 µL in split less mode with an inlet temperature of 250 • C, and helium was used as the carrier gas at a flow rate of 1.0 mL min −1 . The analyses were carried out in programmed mode, the initial oven temperature was held at 45 • C for 2 min, ramped to 200 • C at 5 • C/min, held for 2 min, ramped to 320 • C at 15 • C/min, then held for 3 min. The transfer line, the ion source, and quadrupole analyzer temperatures were 280, 230, and 150 • C, respectively. The ionization mode was the electron impact at 70 eV. The mass spectra plot was acquired using full scan monitoring mode with a mass scan range of m/z 35-450. The acceleration voltage was turned on after a solvent delay of 3 min. The GC analyses were done with an Agilent 7890 GC system. The flame ionization detector (FID) detector temperature was set to 300 • C and the same operational conditions applied to a duplicate of the same column used in GC/MS analyses. The flow rate of carrier gas was adjusted to obtain the same retention times with GC/MS analyses.

Chemical Composition of PSK Volatiles and Extracts
The identification of volatile components was based on the calculated retention indices (RI) and on the comparisons of obtained mass spectra with those of reference compounds, available in the NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA) library. The retention indices were determined in relation to a homologous series of n-alkanes (C7-C30) under the same operating conditions. The relative percentage amounts of the separated compounds were calculated from the integration of the peaks in flame ionization detector (FID) chromatograms.

Pot Experiment
The method of Pn inoculum preparation was as follows. The millets were boiled with water until two-thirds of the seed hulls were ruptured. Filtrated with gauze, aired (with 15% of water content), and placed in flasks for sterilization (121 • C, 20 min). Pn was reactivated on oatmeal agar (OA) medium for seven days. Then, mycelial discs (diameter 5 mm, 3 discs per flask) were inoculated on millet medium and incubated for 14 days at 26 • C.
Soil for the experiment was from the field of Jimo base, Tobacco Institute, Chinese Academy of Agricultural Sciences, type loam, which was sterilized by dry-heat method (160 • C for 5 h). After cooling, the pathogenic soils (PS) were prepared by blending the millet with Pn infection (5 g per kg). PSK were powdered, sieved, and added to the pathogenic soils (2% and 6% PSK content). Soils with no PSK addition served as control. The soils were covered with plastic film for 30 d, each treatment with 15 tobacco seedlings (1 plant and 20 kg of soil per pot)-all with three replicates. The disease index was calculated at 10 d and 20 d, according to Han et al. (2016) [16], with the agronomic characters measured at 30 d. Three plants were randomly selected from each treatment for measurement. Then soils were sampled to analyze chemical indices. The pH value of soil water extract (soil:water, 2.5:1) was measured using the potentiometric method. The electrical conductivity (EC) was measured by the Shanghai Thunder Magnetism DDS-11A Conductivity Meter (Shanghai, China). Soil chemical properties were determined according to the method described by Bao (2005) [17]. The soil organic matter content was determined by oxidation-reduction-titration using furnace digestion. Soil alkali-hydrolyzed nitrogen (AN) content was determined by the alkali-hydrolyzed diffusion method. The NaHCO 3 extraction Mo-Sb anti-spectrophotometry method was used to determine the available phosphorus (AP) in soil, and the total phosphorus (TP) was assayed with the alkali fusion-MO-Sb anti-spectrophotometric method. The available potassium (AK) was measured by the NH 4 OAc (1 M) extraction-flare photometer method. All with three replicates.

Effect of PSK on Soil Microbial Community
DNA of rhizosphere soil [18] was extracted and purified by a DNeasy®PowerSoil®Kit (Qiagen, Hilden, Germany). The total soil DNA was amplified using the bacterial V4 region of 16S rRNA genes (515F and 806R) primer) and fungal ITS1 (internal transcribed spacer) region of 18S rRNA genes (ITS5-1737F, ITS2-2043R). All PCRs were carried out with a Phusion®High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). PCR products were purified with a Gene JET TM Gel Extraction Kit (Thermo Scientific, Waltham, MA, USA). Finally, 250 bp paired-end sequencing and construction of the gene clone libraries were performed at Novogene (Beijing, China) using the Illumina HiSeq 2500 platform. Three plants were randomly selected for sampling in each treatment.
Quality filtering of the raw tags was performed under specific filtering conditions to obtain high-quality clean tags [19], according to the Quantitative Insights Into Microbial Ecology (QIIME) (http://qiime.org/index.html) [20] quality-controlled process. The chimera sequences were removed using the Ultra-fast sequence analysis (UCHIME) (http://www.drive5.com/usearch/manual/uchi-me_ algo.html) [21]. The obtained effective tags were clustered and annotated with Uparse (http://drive5com/ uparse/). Each operational taxonomic unit (OTU) was defined as a cluster of reads with a 97% sequence identity. For each representative bacterial sequence, the Silva Database (http://www.arb-silva.de/) was used based on the Mothur algorithm to annotate taxonomic information, with the fungal sequence annotated by UNITE (v 7.2) Database (https://unite.ut.ee/). Alpha diversity is used to analyze the complexity of species diversity for a sample through observed species, Chao1, Shannon, and ACE. For our samples, all of these indices were calculated with QIIME (Version 1.7.0), and were displayed using R software (Version 2.15.3).
Nonmetric multidimensional scaling (NMDS) of the microbial communities based on OTU composition was carried out using the Bray-Curtis similarity metric in the Past software package, version 2.03 [22]. Analysis of molecular variance (AMOVA) is a nonparametric analysis method, which can test the significance of differences between groups based on the distance between samples. AMOVA analysis used the AMOVA function of Mothur software.
Canonical correspondence analysis (CCA, used CCA function in the vegan package) was used to display the relationship between microbial group (at genus level), soil chemical properties (soil pH, organic matter, hydrolyzable nitrogen), and disease index. CCA was performed using R-2.15.3 for Windows. The Mantel test used the vegan package in R, according to the species matrix and the data matrix of environmental factors provided. The vegdist function was used to transform the distance matrix of the two kinds of data, and then the mantel function was used to analyze the Spearman correlation of the two kinds of matrices to get the r and P values.

Data Analysis
Microsoft Office Excel 2016 was used for data processing; IBM SPSS Statistics version 24.0 was used for variance analysis and correlation analysis. Significance was evaluated at P < 0.05 for all tests. The results of the assays of inhibitory activity, soil properties, and disease index are presented as the means ± standard error of triplicate experiments. The half-maximal inhibitory concentration (IC 50 ) values were calculated using regression analysis of the logarithmic values of component concentrations and probability values of the corresponding inhibition rate. Analysis of variance (ANOVA) and multiple comparisons were performed using Duncan's method. Evaluations with P < 0.05 were considered significant.

Compounds
Retention

Antifungal Effects of PSK Against Phytophthora Nicotianae
Among components of extract, Ethanone-1-(2-hydroxy-4,6-dimethoxyphenyl)-and 9,12-Octadecadienoic acid (Z,Z)-have an obvious inhibitory effect on P. nicotianae, and an antifungal belt is obvious around the dosing hole. But other compounds had no observed effect. The sealed-plate experiment revealed that the volatile components of PSK exhibited inhibitory activity on the growth of P. nicotianae mycelia (Tables 2 and A2). As shown in Figure A1, the mycelia of the control were even and dense ( Figure A1a), while the mycelia treated with PSK grew slowly and loosely ( Figure A1b). The inhibitory effect increased with the increase in the number of prickly ash seeds. The inhibition rate of 6 g/plate PSK was 63.1% (Table A2). In corroboration with the in vitro experiment, the pot experiment also proved that the PSK had an obvious fumigant effect on Pn. Compared to the control, the addition of 2% and 6% PSK decreased the incidence of the disease by 76.47% and 85% after 10 th days of inoculation, respectively ( Figure 1). These results indicated that PSK could be a potential fumigant against tobacco black shank.

Antifungal Effects of PSK Against Phytophthora Nicotianae
Among components of extract, Ethanone-1-(2-hydroxy-4,6-dimethoxyphenyl)-and 9,12-Octadecadienoic acid (Z,Z)-have an obvious inhibitory effect on P. nicotianae, and an antifungal belt is obvious around the dosing hole. But other compounds had no observed effect. The sealed-plate experiment revealed that the volatile components of PSK exhibited inhibitory activity on the growth of P. nicotianae mycelia (Table A2 and Table 2). As shown in Figure A1, the mycelia of the control were even and dense ( Figure A1-a), while the mycelia treated with PSK grew slowly and loosely ( Figure A1-b). The inhibitory effect increased with the increase in the number of prickly ash seeds. The inhibition rate of 6 g/plate PSK was 63.1% (Table A2). In corroboration with the in vitro experiment, the pot experiment also proved that the PSK had an obvious fumigant effect on Pn. Compared to the control, the addition of 2% and 6% PSK decreased the incidence of the disease by 76.47% and 85% after 10 th days of inoculation, respectively ( Figure 1). These results indicated that PSK could be a potential fumigant against tobacco black shank.  The in vitro fumigant activity of PSK can be attributed to its volatile components, especially the higher proportions of monoterpenes. As shown in Table 2, four of the six main constituents (peak area percentage over 6%) tested exhibited strong inhibitory activity against P. nicotianae. Among them, linalool was the most effective one with IC 50 of 18.03 µL/L, followed by piperitone (51.99 µL/L), and sabinene (172.88 µL/L). Limonene was less effective with an IC 50 value of 448.15 µL/L, while myrcene showed no obvious inhibition effect.

Effects of PSK Fumigant on Plant Growth
As shown in Figure 2, the addition of 2% PSK promoted the growth of tobacco, such as higher plant height, root length, and dry weight. This effect may be attributed to soil nutrient improvement. Rahman et al. (2011) [35] reported that the application of two brassica green manure increased soil organic matter. Similarly, Marchetti et al. (2015) [10] found that biofumigant plant materials Brassica carinata and Brassica juncea (containing glucosinolate) improved the soil N availability. In line with the result in previous studies, PSK treatment increased the soil organic matter, hydrolyzable nitrogen, and available potassium ( Table 3). The total phosphorus increased while available phosphorus decreased, suggesting that a large amount of phosphorus is immobilized in the soil. The soil pH was also altered, which decreased with increasing amounts of PSK. Our results suggested that PSK has the potential to be used as not only a fumigant but also as a green manure. However, excessive addition of PSK (≥6%) inhibited plant growth, indicating the existence of allelochemicals.   Values are the means of three replicates ± SD. Values within the same column, followed by different lowercase letters are significantly different (P < 0.05).

Effects of PSK Fumigant on Soil Microbial Community Richness and Diversity
A total of 721,657 fungal sequences (T6, 240,640; T2, 240,320; CK, 240,697) and 721,975 bacterial sequences (T6, 240,684; T2, 240,809; CK, 240,482) were queried after quality filtering, with an average read length of 412-415 bp for bacteria and 230-252 bp for fungi. The rarefaction curves at 3% dissimilarity indicated that the operational taxonomic unit (OTU) number of both fungi and bacteria ( Figure 3) decreased with PSK fumigation. In accordance with this, the richness and diversity values were also reduced, and the reduction showed an obvious dose-effect relationship with PSK. Treatment with 6% PSK resulted in the lowest α-diversity of both bacteria and fungi (Table 4).   Figure 3) decreased with PSK fumigation. In accordance with this, the richness and diversity values were also reduced, and the reduction showed an obvious dose-effect relationship with PSK. Treatment with 6% PSK resulted in the lowest α-diversity of both bacteria and fungi (Table 4).

Effects of PSK Fumigant on Soil Microbial Community Richness and Diversity
Values are the means of three replicates ± SD. Values within the same column, followed by different lowercase letters are significantly different (P < 0.05).
Suppression of soil bacterial or fungal populations by bio-fumigation was reported in several previous studies [36][37][38]. Ma et al. (2018) [39] used Tagetes erecta powder for soil fumigation and found that the diversity and richness index of fungi decreased. Interestingly, our results also indicated that compared to bacteria, the fungi were more susceptible to PSK treatment. The Shannon, Chao1, and ACE indices of bacteria with the addition of 2% PSK were not significantly different from those of the control, while that of fungi were obviously lower compared to that of the control.

Effect of PSK Fumigant on Microbial Structure
The relationship between all soil microbial community compositions was illustrated by NMDS analysis (Figure 4). The NMDS plot showed that three randomly collected replicates from each treatment were usually located closely. The bacterial (Stress = 0.045) and fungal (Stress = 0.001) community of the high PSK (6%), low PSK (2%), and no PSK (control) treatments were well separated from each other, indicating that the composition of the soil microbial community was altered in response to the bio-fumigation treatments.
Agronomy 2020, 10, x FOR PEER REVIEW 10 of 15 Pseudomonas, Azospirillum, Thermomonas, Glutamicibacter (6% PSK), and Dyella (6% PSK) as well as the fungal species Trichoderma, Mortierella, and Aspergillus (6% PSK) ( Figure A2).   According to the result of the Mantel test, the soil parameters that were significantly correlated with the bacterial and fungal community structures were selected for the CCA analysis ( Figure 5). The microbial communities shifted with PSK amendment along CCA1 and CCA2, respectively. Among all the tested environmental variables, organic matter (OC), hydrolyzable nitrogen (N), pH, and disease index (DI) were relatively near CCA1 which explains the 60.22% variation in fungal communities (r = 0.4946, P = 0.001) and 58.15% in the bacterial communities (r = 0.4919, P = 0.001). PSK (6% and 2%) fumigant treatment increased (P < 0.05) the relative abundance of bacterial species Pseudomonas, Azospirillum, Thermomonas, Glutamicibacter (6% PSK), and Dyella (6% PSK) as well as the fungal species Trichoderma, Mortierella, and Aspergillus (6% PSK) ( Figure A2).  To the best of our knowledge, the present study is the first to detail the effects of PSK on soil microbial communities. Therefore, the corroboration of results is not possible. Nevertheless, our findings were partially supported by several related studies. Hu et al. (2015) [40] found that different types of isothiocyanates resulted in significant changes in fungal community composition. Similarly, Weerakoon et al. (2012) [37] reported that modification of the fungal community by the addition of Brassica juncea seed meal might contribute to the long-term suppression of Pythium adappressorium. In line with our results, the increase in Aspergillus [38], Mortierella [40], and Trichoderma [37] were also observed in previous studies, which were believed to be more tolerant to bio-fumigation [41,42]. In line with the results of previous studies, with the application of lower PSK amount, the soil bacterial community was not severely affected [38,40]. Hollister et al. (2012) [36] found that bacteria related to fungal disease control (such as Bacillus, Pseudomonas, and Streptomyces) increased with the application of a glucosinolate-containing brassicaceous oilseed meal (Brassica juncea). PSK fumigant also increased the relative abundance of bacterial genera, which were reported to be associated with plant growth promotion or disease control [39,43,44]. Based on these results, we hypothesized that the microbial community shift following PSK application could be one of the important mechanisms for disease suppression. To the best of our knowledge, the present study is the first to detail the effects of PSK on soil microbial communities. Therefore, the corroboration of results is not possible. Nevertheless, our findings were partially supported by several related studies. Hu et al. (2015) [40] found that different types of isothiocyanates resulted in significant changes in fungal community composition. Similarly, Weerakoon et al. (2012) [37] reported that modification of the fungal community by the addition of Brassica juncea seed meal might contribute to the long-term suppression of Pythium adappressorium. In line with our results, the increase in Aspergillus [38], Mortierella [40], and Trichoderma [37] were also observed in previous studies, which were believed to be more tolerant to bio-fumigation [41,42]. In line with the results of previous studies, with the application of lower PSK amount, the soil bacterial community was not severely affected [38,40]. Hollister et al. (2012) [36] found that bacteria related to fungal disease control (such as Bacillus, Pseudomonas, and Streptomyces) increased with the application of a glucosinolate-containing brassicaceous oilseed meal (Brassica juncea). PSK fumigant also increased the relative abundance of bacterial genera, which were reported to be associated with plant growth promotion or disease control [39,43,44]. Based on these results, we hypothesized that the microbial community shift following PSK application could be one of the important mechanisms for disease suppression.

Conclusions
Findings in the current study clearly demonstrated that the application of appropriate PSK (2%) is capable of effectively controlling tobacco black shank. The fumigant effect can be attributed to the volatiles released by PSK, such as linalool, piperitone, sabinene, and limonene. Moreover, PSK fumigation also significantly promoted tobacco growth and improved soil fertility (total P, hydrolysable N, and available K), exhibiting the effect of a fungicide and fertilizer. Interestingly, the shifts in microbial community structure showed increased proportions of bacterial taxa associated with pathogen suppression and plant growth promotion, suggesting that PSK can be applied in combination with certain biocontrol microbes.
From this study, we concluded that PSK could be a new bio-fumigation material against plant soilborne pathogens. However, long-term, field-scale studies are needed to evaluate the fumigation effect of PSK.

Appendix A
From this study, we concluded that PSK could be a new bio-fumigation material against plant soilborne pathogens. However, long-term, field-scale studies are needed to evaluate the fumigation effect of PSK.    Figure A2. Heatmap at genus level of soil bacterial (a) and fungal (b). T2, 2% of PSK amendment; T6, 6% of PSK amendment; CK, control with no PSK amendment. Table A1. Composition of the extract of prickly ash seed kernel by gas chromatography coupled with flame ionization detection.