Diffusible and Volatile Antifungal Compounds Produced by Pseudomonas chlororaphis subsp. aurantiaca ST-TJ4 against Various Phytopathogenic Fungi CURRENT STATUS: POSTED

Plant growth-promoting rhizobacteria can potentially be used as an alternative strategy to control plant disease. In this study, strain ST-TJ4 isolated from the rhizosphere soil of a healthy poplar was found to have strong antifungal activity against 11 phytopathogenic fungi in agriculture and forestry. Strain ST-TJ4 was identified as Pseudomonas chlororaphis subsp. aurantiaca based on 16S rDNA sequences. The bacterium can produce siderophores, cellulase, and protease, and has genes involved in the synthesis of phenazine, 1-phenazinecarboxylic acid, pyrrolnitrin, and hydrogen cyanide. Moreover, the volatile compounds released by strain ST-TJ4 can inhibit the mycelial growth of plant pathogenic fungi more than diffusible substances can. Based on volatile compound profiles of strain ST-TJ4 obtained from headspace collection and GC-MS/MS analysis, 1-undecene was identified. In summary, the results suggested that P. chlororaphis subsp. aurantiaca ST-TJ4 can be used as a biocontrol agent for various plant diseases caused by phytopathogenic fungi.


Introduction
Severe crop loss remains inevitable due to plant diseases, particularly those caused by pathogenic fungi, which are responsible for an estimated 70-80% of plant diseases fungi. Furthermore, the species and numbers of plant fungal pathogens in the rhizosphere change with environmental conditions and evolution, which increases the difficulty of controlling plant diseases (Pieterse et al., 2016). Facing the severe threat to global crop security caused by plant diseases, it is important to develop environmentally friendly and highly effective fungicides against plant pathogens.
Compared with application of synthetic chemical fungicides, the use of microorganisms and their metabolites is a promising and environmentally friendly alternative for the prevention and control of plant diseases (Sahni et al., 2008). Biological control factors such as Bacillus, Pseudomonas, Burkholderia, and Paenibacillus spp. play important roles in inhibiting pathogens Fan et al., 2018;Zhang et al., 2019;Wang et al., 2019). Bacillus species can produce a wide variety of antimicrobial compounds with different chemical structures, including lipopeptides, polyketides and bacteriocins (Olfa et al., 2015). The siderophore-mediated competition for iron gives beneficial microbes a competitive advantage in suppressing the proliferation and root colonization of plant pathogens (Maindad et al., 2014;Kong et al., 2019).
In addition, microbial volatile organic compounds (VOCs) have attracted more attention because they can spread over long distances, mediating indirect contact interactions between organisms. Low concentrations of VOCs can be sensed, so they can directly inhibit the growth of pathogenic fungi and induce systemic resistance in plants (Cordero et al., 2014;Velivelli et al., 2015). The VOCs produced by Bacillus can inhibit mycelial growth of Alternaria solani and Botrytis cinerea, which cause early blight and grey mold, respectively . They can also control Ceratocystis fimbriata in postharvest sweet potatoes (Zhang et al., 2010). It is suggested that the application of microbial volatiles has great potential in plant diseases.
Current research regarding biocontrol bacteria is mainly focused on crop applications and rarely considers forest diseases. In preliminary analyses prior to this study, we screened a bacterial strain with high siderophore production from the poplar rhizosphere; however, it remains unclear whether this strain has antagonistic effects on fungi causing forest diseases or produces additional antagonistic substances. Therefore, in this study, we identified diffusible substances and VOCs produced by this strain to determine whether they may be applied as antagonistic substances. We also determined the taxonomic status of this strain via morphological identification and molecular biology. The results of this study may provide a new and effective antagonistic bacterial strain for the biological control of forest fungal diseases, and lay a foundation for molecular analyses of the bacteriostatic mechanism of this strain.
These fungal plant pathogens were preserved in the Forest Pathology Laboratory of Nanjing Forestry University. The isolates were maintained on Potato dextrose agar (PDA) plates at 25 °C before use. 5 2.2. In vitro antifungal activity Using in vitro dual-culture analysis, strain ST-TJ4 was subjected to in vitro antifungal activity assays against the above 11 phytopathogenic fungi. PDA medium was used as basal medium. A 0.6-cm-diameter plug containing mycelia was taken from 7-day-old target fungi and placed at the center of PDA dual plates. Single ST-TJ4 colonies were patched about 2.5 cm from the fungus. After 3-7 days, the width of the inhibition zone between the bacterial colony and fungal pathogen was measured. Each treatment was replicated four times. The experiment was also repeated twice.

Analysis of the antagonistic substances in vitro
Siderophore production was determined using the Chrome Azurol S (CAS) assay (Schwyn and Neilands 1987). The method proposed by Han et al. (2015) was modified as follows to examine the chitinase and protease activities of the metabolites from ST-TJ4. Colloidal chitin and skim milk powder (SMP) were added to PDA plates to final concentrations of 1 mg/mL and 3% (w/v), respectively. To examine the glucanase activity of ST-TJ4, the method described by Suryadi et al. (2014) was used, adding yeast β-glucan at a final concentration of 0.2% (w/v) to PDA plates. Individual ST-TJ4 colonies were cultured in liquid LB medium overnight at 28 °C with shaking. When the OD 600 was 0.6, 2 µL of the culture was plated on PDA plates containing colloidal chitin, SMK, or β-glucan and placed in an incubator at 28 °C (Huang et al., 2017). Three days later, the PDA plates containing colloidal chitin and SMP were observed and evaluated for the presence of a clear zone surrounding the bacterial colonies caused by chitinase and protease degradation. The plates containing β-glucan were stained, and glucanase activity was observed using the method of Suryadi et al. (2014).

Detection of genes encoding antibiotics and HCN in ST-TJ4
Total DNA was isolated from ST-TJ4 cells by the cetyltrimethylammonium bromide (CTAB) method (Ausubel et al., 2001). Then, polymerase chain reaction (PCR) assays were used to detect the phzCD, phz, and prnC genes according to protocols described in Raaijmakers et al. (1997) and Hu et al. (2014). The hcnAB gene was detected as previously described (Svercel et al., 2007). The primers used in the experiment are shown as Table 1: Table 1 Oligonucleotide primers used in this study

Identification of strain ST-TJ4
The biochemical tests were completed following the standard methods in Bergey's Manual of Determinative Bacteriology (Holt et al., 1994). For molecular identification of ST-TJ4, bacterial genomic DNA was extracted and purified using the CTAB method (Ausubel et al., 2001). The 16S rRNA gene was PCR amplified using the universal primers 27f (5'AGAGTTTGATCATGGCTCAG3') and 1492R (5'TACGGYTACCTTGTTACGACTT3'). Sequence homology searches were performed using the online blast search engine in EzTaxon-eDatabase-EzBioCloud (http://www.ezbiocloud.net/eztaxon). The sequences were aligned and a phylogenetic tree constructed using Mega 7.0 Beta (Tamura, et al., 2013). A tree was obtained using the neighbor-joining method based on the Tamura 3-parameter model with the 'gaps complete deletion' option, and the tree was drawn with branch lengths measured in number of substitutions per site. The interior-branch test using 1000 bootstrap replications was used to assess the relative stability of the branches.

Statistical analysis
Statistical significance was determined using SPSS 17.0, with one-way analysis of variance sapinea, but only weakly affected that of C. chrysosperma, C. tropicale and R. solani ( Fig. 1).

Siderophore and cell wall degradation enzyme activity of ST-TJ4
To examine the antimicrobial substances produced by ST-TJ4, including cellulase, chitinase, protease, and glucanase, ST-TJ4 was inoculated on PDA plates containing cellulose, colloidal chitin, SMP, or β-glucan. After 3 days, an obvious hydrolysis zone was observed on the agar plates containing cellulose and SMP, but not on those containing chitin or β-glucan (Fig. 2). These results indicated that ST-TJ4 has strong siderophore, cellulase, and proteolytic activities, but no chitinolytic or glucanolytic activity.

Detection of antibiotic and HCN encoding genes in ST-TJ4
We tested strain ST-TJ4 for the presence of operons for the biosynthesis of the antibiotics phenazine-1-carboxylic acid (PCA), phenazine (PHZ), pyrrolnitrin (PRN), and hydrogen cyanide (HCN) by PCR using specific primers. A fragment of the predicted size for each of these compounds was observed from the DNA of the reference Pseudomonas strains, which produces these compounds. Fragments of the predicted sizes for PCA (~ 1150 bp), PHZ (~ 1408 bp), PRN (~ 786 bp), and HCN (~ 570 bp) were amplified from strain ST-TJ4 DNA (Fig. 3).

The antifungal spectrum of ST-TJ4 VOCs
The two-sealed-base-plates method was used to determine the antifungal spectrum of ST-

GC-MS/MS analysis of VOCs produced by ST-TJ4
Volatiles from strain ST-TJ4 were collected in a SPME syringe and analyzed with a GC-MS/MS system. The same volatiles as in the LB medium and substances with relative contents less than 0.5% were filtered out. This revealed very clear separation between the control and strain ST-TJ4, as indicated in Fig. 5. Five different VOCs emitted by strain ST-TJ4 that were absent in the control were identified. The most abundant volatile produced by strain ST-TJ4 was 1-undecene, which had a large peak (75.97%) at a retention time (RT) of 8.65 minutes (Table 3). The other compounds were l-Ala-l-Ala-l-Ala (1.42%), octamethylcyclotetrasiloxane (1.14%), 4-hydroxybenzoic acid (1.06%), and phosphonoacetic acid (0.54%).

Identification of strain ST-TJ4
Colonies of ST-TJ4 on LB plates appeared thin, flat, orange, opaque, round with smooth edges, and approximately 1.2-3 mm in diameter (Fig. 6a). The oxygen demand test showed that ST-TJ4 is an aerobic bacterium. As shown in the picture, it grows well outside the cover glass, but hardly grows under the cover glass (Fig. 6b). Gram staining showed that ST-TJ4 is a Gram-negative bacterium (Fig. 6c). The phylogenetic analysis showed that the 16S rRNA nucleotide sequence of strain ST-TJ4 shared high similarity (89%) with the reference species Pseudomonas chlororaphis subsp. aurantiaca (Fig. 7). Based on the morphological characteristics and phylogenetic analysis, strain ST-TJ4 was identified as Pseudomonas chlororaphis subsp. aurantiaca.

Discussion
One of the biggest ecological challenges facing microbiologists and plant pathologists in the near future is the development of environmentally friendly alternatives to chemical pesticides for combating plant diseases (Roquigny et al., 2018). The use of beneficial microorganisms is one of the most promising methods for safe, rational crop-management practices (Ongena and Jacques, 2008). Thus, we attempted to isolate antagonistic bacteria with strong antifungal activity against 11 phytopathogenic fungi. We found that strain ST-TJ4 inhibited the mycelial growth of various plant pathogenic fungi. This suggested that strain ST-TJ4 has a wide fungicide spectrum and could produce antifungals. Based on the morphological characteristics and phylogenetic analysis, strain ST-TJ4 was identified as P.
Bacteria can produce cell wall-degrading enzymes and secondary metabolites to hinder the growth of other microorganisms. Fungal cell walls play important roles during cell division and growth and in maintaining hyphal morphology and adaptation to the environment (Adams, 2004;Cabib et al., 2001). In pathogenic fungi, the ability to maintain cell wall integrity is critical for establishing diseases in the host (Jeon et al., 2008). The fungal cell wall is a matrix of three main components: chitin, glucans, and proteins (Webster and Weber, 2007;Lenardon et al., 2010;Pareek et al., 2014). The destruction of these components usually leads to changes in morphology and loss of the biological function of fungal hyphae. In this study, ST-TJ4 had strong cellulase and protease activities on culture plates containing β-glucan and SMP, but no hydrolytic activity on the plates containing colloidal chitin. This indicates that ST-TJ4 has the potential to degrade the cellulose and proteins of fungal hyphae.
Antibiotics are toxins produced by microbes that can kill other microorganisms (Rovera et al., 2014;Chen et al., 2018). Antibiosis is one of the most studied mechanisms of biological control and a selection trait to consider when screening potential biological control agents (BCAs) (Raio et al., 2011;Filion et al., 2018). The production of pigments in a medium indicates that bacteria are producing metabolites. We performed experiments to detect biosynthetic genes implicated in the production of antifungal compounds in P. reported to have antibacterial and antifungal activity (Lin et al., 2014) and was found in the present study. To our knowledge, this is the first report on the emission of 1-undecene and octamethylcyclotetrasiloxane by P. chlororaphis subsp. aurantiaca, which has a broad bacteriostatic spectrum.
Interestingly, the diffusible substances produced by ST-TJ4 showed weak antagonistic effects against C. chrysosperma, R. solani, and C. tropicale, whereas its volatiles almost completely inhibited the growth of these three pathogenic fungi. Because we used LB medium to culture bacteria in the two-sealed-base-plates method, while strain ST-TJ4 was streaked onto PDA plates in the dual-culture experiment. It is possible that the inoculation strategy led to different antagonistic effects; that is, the inhibition rate of spreading small cells may not have been as great as that of the spread cells exposed to larger surfaces of the medium, which would allow the single cells to obtain more nutrients (Rajer et al., 2017

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Competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.  In vitro antifungal activity of ST-TJ4 against various phytopathogenic fungi in dual-culture assays.  The antifungal spectrum of strain ST-TJ4 VOCs. The right side is treated with VOCs, and the left side is untreated.

Figure 7
Phylogenetic tree derived from the 16S rDNA sequence of Pseudomonas chlororaphis subsp. aurantiaca strain ST-TJ4. The tree was constructed using the neighbor-joining method with 1000 bootstrap resamplings using MEGA 7.0.