Identification of the Biosynthetic Gene Cluster for the anti-MRSA Lysocins through Gene Cluster Activation Using Strong Promoters of Housekeeping Genes and Production of New Analogs in Lysobacter sp. 3655.

The Gram-negative gliding bacteria Lysobacter represent a new and rich source for bioactive natural products. In an effort to discover new antibiotics, we found a cryptic biosynthetic gene cluster (BGC) in Lysobacter sp. 3655 that shared a high similarity with the putative lysocin BGC identified in silico previously from Lysobacter sp. RH2180-5. Lysocins are cyclic lipodepsipeptides with potent activity against MRSA (methicillin-resistant Staphylococcus aureus) using a novel mode of action, but the lysocin BGC had not been experimentally verified so far. Using an activity-guided screening, we isolated the main antibiotic compound and confirmed it to be lysocin E. However, the putative lysocin BGC was barely transcribed in the wild type, in which lysocins were produced only in specific conditions and in a negligible amount. To activate the putative lysocin BGC, we screened for strongly transcribed housekeeping genes in strain 3655 and found several powerful promoters. Upon engineering the promoters into the BGC, the lysocin gene transcription was significantly enhanced and the lysocin yield was markedly increased. With readily detectable lysocins production in the engineered strains, we showed that lysocin production was abolished in the gene deletion mutant and then restored in the complementary strain, even when grown in conditions that did not support the wild type for lysocin production. Moreover, the engineered strain produced multiple new lysocin congeners. The determination of the lysocin BGC and the Lysobacter promoters will facilitate the ongoing efforts for yield improvement and new antibiotic biosynthesis using synthetic biology strategies.

Control of Wheat Fusarium Head Blight by Heat-Stable Antifungal Factor (HSAF) from Lysobacter enzymogenes.

Heat-stable antifungal factor (HSAF), which belongs to the polycyclic tetramate macrolactam family, was isolated from Lysobacter enzymogenes fermentations and exhibited inhibitory activities against a wide range of fungal pathogens. In this study, the antifungal activity of HSAF against Fusarium graminearum in vitro and in vivo was investigated. A total of 50% of mycelial growth of F. graminearum was suppressed with 4.1 µg/ml of HSAF (EC50 value). HSAF treatment resulted in abnormal morphology of the hyphae, such as curling, apical swelling, and depolarized growth. Furthermore, HSAF adequately inhibited conidial germination and conidiation of F. graminearum with an inhibition rate of 100% when 1 and 6 µg/ml of HSAF were applied, respectively. HSAF caused ultrastructural changes of F. graminearum, including cell wall thickening and plasmolysis. Moreover, the application of HSAF significantly controlled Fusarium head blight in wheat caused by F. graminearum in the field. Overall, these results indicate that HSAF has potential for development as a fungicide against F. graminearum.

Protecting books from mould damage by decreasing paper bioreceptivity to fungal attack using decoloured cell-free supernatant of Lysobacter enzymogenes C3.

AIMS: To evaluate whether decoloured cell-free supernatant of Lysobacter enzymogenes C3 can decrease paper bioreceptivity to fungal attack. METHODS AND RESULTS: To prepare colourless C3 supernatant, single-factor design and uniform design were applied. The optimum conditions with high decolouration degree and low antifungal activity loss were achieved as follows: carbon granule content 1·6% (M/V), temperature 27°C, decolouring time 1·2 h and pH 8·0. An agar plate bioassay was used to assess the antifungal activity of the decoloured supernatant against the fungal isolates obtained from contaminated books, and strong suppression was observed. Small-sacle laboratory test was further introduced, in which common book papers were artificially inoculated with the fungal isolates, and then sprayed uniformly with decoloured supernatant or water. The results showed that, after treatment, the paper showed a significantly low extent of fungal colonization and high tensile strength, and maintained the same colour before and after treatment. CONCLUSION: These results suggest that the decoloured C3 supernatant inhibits fungal growth on types of paper commonly used in books. SIGNIFICANCE AND IMPACT OF THE STUDY: Decoloured C3 supernatant could be used as a preventive agent to protect books and other paper-based items against fungal growth.

Proposal of Lysobacter pythonis sp. nov. isolated from royal pythons (Python regius).

The bacterial strains 4284/11T and 812/17 isolated from the respiratory tract of two royal pythons in 2011 and 2017, respectively were subjected to taxonomic characterization. The 16S rRNA gene sequences of the two strains were identical and showed highest sequence similarities to Lysobacter tolerans UM1T (97.2%) and Luteimonas aestuarii DSM 19680T (96.7 %). The two strains were identical in the sequences of the 16S-23S rRNA internal transcribed spacer (ITS) and partial groEL gene sequences and almost identical in genomic fingerprints. In the ITS sequence Ly. tolerans DSM 28473T and in the groEL nucleotide sequence Luteimonas mephitis DSM 12574T showed the highest similarity. In silico DDH analyses using genome sequence based ANIb and gANI similarity coefficients demonstrated that strain 4284/11T represents a novel species and revealed Ly. tolerans UM1T as the next relative (ANIb = 76.2 %, gANI = 78.0 %). Based on the topology of a core gene phylogeny strain 4284/11T could be assigned to the genus Lysobacter. Chemotaxonomic characteristics including polyamine pattern, quinone system, polar lipid profile and fatty acid profile were in accordance with the characteristics of the genera Lysobacter and Luteimonas. Strains 4284/11T and 812/17 could be differentiated from the type strains of the most closely related species by several physiological tests. In conclusion we are here proposing the novel species Lysobacter pythonis sp. nov. The type strain is 4284/11T (= CCM 8829T = CCUG 72164T = LMG 30630T) and strain 812/17 (CCM 8830) is a second strain of this species.

Antifungal Metabolite p-Aminobenzoic Acid (pABA): Mechanism of Action and Efficacy for the Biocontrol of Pear Bitter Rot Disease.

Colletotrichum fructicola, a fungal pathogen that causes bitter rot disease in pears, has recently emerged in Eastern Asia and caused enormous economic losses and crop penalties. For this reason, new strategies for the management of bitter rot disease are greatly needed and can have a great impact on the field. In this regard, our research group recently reported that p-aminobenzoic acid (pABA), which was found in the secretions of rhizobacterium Lysobacter antibioticus OH13, showed a broad spectrum of antifungal activities. Following this project, the antifungal mode of action of pABA has been elucidated in this work indicating that pABA affects the fungal cell cycle of C. fructicola by inhibiting septation during cell division. pABA stability and diffusion screening revealed that pABA degrades after 15 days and is able to cross the pear skin into the external parts of the mesocarp. In vivo studies demonstrated that pABA shows high curative ability against the infection of C. fructicola in pears. To show the efficacy of OH13 for the biocontrol of bitter rot disease, cultures of OH13 containing 379.4 mg/L pABA were sprayed on inoculated pears, significantly reducing the symptoms of the pathogen.

Efficient production of heat-stable antifungal factor through integrating statistical optimization with a two-stage temperature control strategy in Lysobacter enzymogenes OH11.

BACKGROUND: Heat-stable antifungal factor (HSAF) is a newly identified broad-spectrum antifungal antibiotic from the biocontrol agent Lysobacter enzymogenes and is regarded as a potential biological pesticide, due to its novel mode of action. However, the production level of HSAF is quite low, and little research has reported on the fermentation process involved, representing huge obstacles for large-scale industrial production. RESULTS: Medium capacity, culture temperature, and fermentation time were identified as the most significant factors affecting the production of HSAF and employed for further optimization through statistical methods. Based on the analysis of kinetic parameters at different temperatures, a novel two-stage temperature control strategy was developed to improve HSAF production, in which the temperature was increased to 32 °C during the first 12 h and then switched to 26 °C until the end of fermentation. Using this strategy, the maximum HSAF production reached 440.26 ± 16.14 mg L- 1, increased by 9.93% than that of the best results from single-temperature fermentation. Moreover, the fermentation time was shortened from 58 h to 54 h, resulting in the enhancement of HSAF productivity (17.95%) and yield (9.93%). CONCLUSIONS: This study provides a simple and efficient method for producing HSAF that could be feasibly applied to the industrial-scale production of HSAF.

Vesicular Delivery of the Antifungal Antibiotics of Lysobacter enzymogenes C3.

Lysobacter enzymogenes C3 is a predatory strain of Gram-negative gliding bacteria that produces antifungal antibiotics by the polyketide synthetic pathway. Outer membrane vesicles (OMV) are formed as a stress response and can deliver virulence factors to host cells. The production of OMV by C3 and their role in antifungal activity are reported here. Vesicles in the range of 130 to 150 nm in diameter were discovered in the cell-free supernatants of C3 cultures. These OMV contain molecules characteristic of bacterial outer membranes, such as lipopolysaccharide and phospholipids. In addition, they contain chitinase activity and essentially all of the heat-stable antifungal activity in cell supernatants. We show here that C3 OMV can directly inhibit growth of the yeast Saccharomyces cerevisiae as well as that of the filamentous fungus Fusarium subglutinans The activity is dependent on physical contact between OMV and the cells. Furthermore, fluorescent lipid labeling of C3 OMV demonstrated transfer of the membrane-associated probe to yeast cells, suggesting the existence of a mechanism of delivery for membrane-associated molecules. Mass spectrometric analysis of C3 OMV extracts indicates the presence of molecules with molecular weights identical to some of the previously identified antifungal products of C3. These data together suggest that OMV act as an important remote mobile component of predation by LysobacterIMPORTANCE The data presented here suggest a newly discovered function of outer membrane vesicles (OMV) that are produced from the outer membrane of the bacterial species Lysobacter enzymogenes strain C3. We show that these OMV can be released from the surface of the cells to deliver antibiotics to target fungal organisms as a mechanism of killing or growth inhibition. Understanding the role of OMV in antibiotic delivery can generally lead to improved strategies for dealing with antibiotic-resistant organisms. These results also add to the evidence that some bacterially produced antibiotics can be discovered and purified using methods designed for isolation of nanoscale vesicles. Information on these systems can lead to better identification of active molecules or design of delivery vehicles for these molecules.

Biosynthesis of the Polycyclic System in the Antifungal HSAF and Analogues from Lysobacter enzymogenes.

The biocontrol agent Lysobacter enzymogenes produces polycyclic tetramate macrolactams (PoTeMs), including the antifungal HSAF. To elucidate the biosynthesis of the cyclic systems, we identified eleven HSAF precursors/analogues with zero, one, two, or three rings through heterologous expression of the HSAF gene cluster. A series of combinatorial gene expression and deletion experiments showed that OX3 is the "gatekeeper" responsible for the formation of the first 5-membered ring from lysobacterene A, OX1 and OX2 are responsible for formation of the second ring but with different selectivity, and OX4 is responsible for formation of the 6-membered ring. In vitro experiments showed that OX4 is an NADPH-dependent enzyme that catalyzes the reductive cyclization of 3-dehydroxy alteramide C to form 3-dehydroxy HSAF. Thus, the multiplicity of OX genes is the basis for the structural diversity of the HSAF family, which is the only characterized PoTeM cluster that involves four redox enzymes in the formation of the cyclic system.

Lysobacter tongrenensis sp. nov., isolated from soil of a manganese factory.

A Gram-staining negative, aerobic, non-motile, rod-shaped bacterial strain, designated YS-37T, was isolated from soil in a manganese factory, People's Republic of China. Based on16S rRNA gene sequence analysis, strain YS-37T was most closely related to Lysobacter pocheonensis Gsoil 193T (97.0%), Lysobacter dokdonensis DS-58T (96.0%) and Lysobacter daecheongensis Dae08T (95.8%) and grouped together with L. pocheonensis Gsoil 193T and Lysobacter dokdonensis DS-58T. The DNA-DNA hybridization value between strain YS-37T and L. pocheonensis KCTC 12624T was 43.3% (± 1). The major respiratory quinone of strain YS-37T was ubiquinone-8, and the polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipid, phosphatidylmethylethaolamine and two unknown lipids. Its major cellular fatty acids (> 5%) were iso-C15:0, iso-C17:1 ω9c, iso-C16:0, iso-C11:0 3-OH and iso-C11:0 and the G + C content of the genomic DNA was 67.1 mol%. Strain YS-37T also showed some biophysical and biochemical differences with the related strains, especially in hydrolysis of casein. The results demonstrated that strain YS-37T belongs to genus Lysobacter and represents a novel Lysobacter species for which the name Lysobacter tongrenensis sp. nov. is proposed. The type strain is YS-37T (= CCTCC AB 2016052T = KCTC 52206T).

Production of Antifungal p-Aminobenzoic Acid in Lysobacter antibioticus OH13.

Among Lysobacter species, Lysobacter antibioticus has been demonstrated to be an interesting source of antimicrobial metabolites for the biocontrol of plant diseases. Although the antibacterial activity was attributed to N-oxide phenazines, the active compounds involved in the antifungal function remained unknown. In this work, an antifungal compound was isolated and identified as p-aminobenzoic acid (pABA). Antifungal activity screening revealed that pABA shows activity against a number of plant pathogens. The genes involved in the synthetic route of this compound in OH13 were identified. Further, the production of pABA was optimized by modification of the carbon source using engineered L. antibioticus OH13 strains.

Visualizing small differences using subtractive chromatographic analysis.

Subtraction of chromatograms coming from two different samples collected under identical conditions can highlight small variations, serving as a useful tool for visualizing differences between experimental and control groups. While the basis for this general approach has been known for decades, the technique is seldom used in modern chromatographic analysis. We report an investigation into the application of subtractive chromatographic analysis in several areas of pharmaceutical research where detection of small differences between samples is important. Our investigation found that elimination of artifacts caused by peak misalignment was often necessary, especially for extremely sharp chromatographic peaks obtained in rapid injection MISER chromatography. Alignment of individual peaks prior to subtraction, combined with fast detector sampling rates, or data interpolation in cases where this is not possible, was found to afford convenient visualization of small differences (∼1%) among samples, suggesting potential utility in high throughput screening of process adsorbents or other applications in pharmaceutical research and development.

Lysobacter species: a potential source of novel antibiotics.

Infectious diseases threaten global health due to the ability of microbes to acquire resistance against clinically used antibiotics. Continuous discovery of antibiotics with a novel mode of action is thus required. Actinomycetes and fungi are currently the major sources of antibiotics, but the decreasing rate of discovery of novel antibiotics suggests that the focus should be changed to previously untapped groups of microbes. Lysobacter species have a genome size of ~6 Mb with a relatively high G + C content of 61-70 % and are characterized by their ability to produce peptides that damage the cell walls or membranes of other microbes. Genome sequence analysis revealed that each Lysobacter species has gene clusters for the production of 12-16 secondary metabolites, most of which are peptides, thus making them 'peptide production specialists'. Given that the number of antibiotics isolated is much lower than the number of gene clusters harbored, further intensive studies of Lysobacter are likely to unearth novel antibiotics with profound biomedical applications. In this review, we summarize the structural diversity, activity and biosynthesis of lysobacterial antibiotics and highlight the importance of Lysobacter species for antibiotic production.

Biogenesis of Lysobacter sp. XL1 vesicles.

The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained.

Bioactive Polycyclic Tetramate Macrolactams from Lysobacter enzymogenes and Their Absolute Configurations by Theoretical ECD Calculations.

Two new polycyclic tetramate macrolactams, lysobacteramides A (1) and B (2), together with HSAF (heat-stable antifungal factor, 3), 3-dehydroxy HSAF (4), and alteramide A (5) were isolated from a culture of Lysobacter enzymogenes C3 in nutrient yeast glycerol medium. Their structures were determined by MS and extensive NMR analysis. The absolute configurations of 1-5 were assigned by theoretical calculations of their ECD spectra. Although HSAF and analogues were reported from several microorganisms, their absolute configurations had not been established. The isolation and the absolute configurations of these compounds revealed new insights into the biosynthetic mechanism for formation of the polycycles. Compounds 1-4 exhibited cytotoxic activity against human carcinoma A549, HepG2, and MCF-7 cells with IC50 values ranging from 0.26 to 10.3 µM. Compounds 2 and 3 showed antifungal activity against Fusarium verticillioides with IC50 value of 47.9 and 6.90 µg/mL, respectively.

Characterization of water-soluble dark-brown pigment from Antarctic bacterium, Lysobacter oligotrophicus.

Lysobacter oligotrophicus strain 107-E2(T) isolated from Antarctica produces dark-brown colored water-soluble pigment, in addition to hydrolases and lytic enzymes. The production of pigment is a common characteristic among members of the genus Lysobacter, but the identity of the pigments has been unknown. In this study, we identified the pigment from L. oligotrophicus as melanin pigment (Lo-melanin) by chemical and spectroscopic analyses. Although melanin is generally insoluble in both aqueous and organic solvents, the results in this study revealed that Lo-melanin shows water-solubility by means of the added polysaccharide chain. Lo-melanin production of L. oligotrophicus was increased by ultraviolet (UV) exposure, and survival rate of Escherichia coli under UV-irradiated condition was increased by the addition of Lo-melanin to the medium.

Lysobacter capsici AZ78 produces cyclo(L-Pro-L-Tyr), a 2,5-diketopiperazine with toxic activity against sporangia of Phytophthora infestans and Plasmopara viticola.

AIMS: To investigate low molecular weight compounds produced in vitro by Lysobacter capsici AZ78 and their toxic activity against sporangia of plant pathogenic oomycetes. METHODS AND RESULTS: Assays carried out in vitro showed that L. capsici AZ78 drastically inhibits the growth of plant pathogenic oomycetes. Accordingly, the preventive application of culture filtrates of L. capsici AZ78 on grapevine and tomato plants reduced the infections, respectively, caused by Plasmopara (Pl.) viticola and Phytophthora infestans. The subsequent chemical analysis of the culture filtrates of L. capsici AZ78 by spectroscopic (essentially 1D and 2D (1)H NMR and (13)C NMR and ESI MS spectra) and optical methods led to the identification of the 2,5-diketopiperazine cyclo(L-Pro-L-Tyr) that inhibited the development of P. infestans sporangia in vitro and on tomato leaves. Furthermore, a genomic region with high sequence identity with genes coding for a hybrid polyketide synthase and nonribosomal peptide synthetase was detected in L. capsici AZ78. CONCLUSIONS: Lysobacter capsici AZ78 produces cyclo(L-Pro-L-Tyr) in vitro that was effective in killing the sporangia of P. infestans and Pl. viticola in vitro. Moreover, this low molecular weight compound prevents the occurrence of late blight lesions when applied on tomato leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of L. capsici AZ78 cells or its own culture filtrates effectively controls both P. infestans and Pl. viticola. Cyclo(L-Pro-L-Tyr) produced by L. capsici AZ78 is toxic against sporangia of both these oomycetes. These data enforce the potential in the use of Lysobacter members for the control of plant pathogenic oomycetes and provide the basis for the development of new low-impact fungicides based on cyclo(L-Pro-L-Tyr).

Involvement of both PKS and NRPS in antibacterial activity in Lysobacter enzymogenes OH11.

Polyketides and nonribosomal peptides represent two large families of natural products (NPs) with diverse structures and important functions. They are synthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS), respectively. Lysobacter enzymogenes is emerging as a novel biocontrol agent against pathogens of crop plants and a new source of bioactive NPs, such as antibacterial antibiotic WAP-8294A2 and antifungal antibiotic HSAF. Genome survey of strain OH11, a Chinese L. enzymogenes isolate, detected four novel PKS, NRPS or hybrid gene clusters, designed as cluster A to D. We further individually mutated five genes (PKS or NRPS) located in these four gene clusters and showed that a PKS gene in cluster A and an NRPS gene in cluster D were involved in the antibacterial activity via a WAP-8294A2 dependent way. The data also showed that none of the five genes was associated with antifungal activity and the regulation of HSAF biosynthesis. Our results reveal the unusual regulatory role of these PKS and NRPS genes that were discovered from genome mining in L. enzymogenes.

Bioactive natural products from Lysobacter.

The gliding Gram-negative Lysobacter bacteria are emerging as a promising source of new bioactive natural products. These ubiquitous freshwater and soil microorganisms are fast growing, simple to use and maintain, and genetically amenable for biosynthetic engineering. This Highlight reviews a group of biologically active and structurally distinct natural products from the genus Lysobacter, with a focus on their biosyntheses. Although Lysobacter sp. are known as prolific producers of bioactive natural products, detailed molecular mechanistic studies of their enzymatic assembly have been surprisingly scarce. We hope to provide a snapshot of the important work done on the lysobacterial natural products and to provide useful information for future biosynthetic engineering of novel antibiotics in Lysobacter.

Observing the invisible through imaging mass spectrometry, a window into the metabolic exchange patterns of microbes.

Many microbes can be cultured as single-species communities. Often, these colonies are controlled and maintained via the secretion of metabolites. Such metabolites have been an invaluable resource for the discovery of therapeutics (e.g. penicillin, taxol, rapamycin, epothilone). In this article, written for a special issue on imaging mass spectrometry, we show that MALDI-imaging mass spectrometry can be adapted to observe, in a spatial manner, the metabolic exchange patterns of a diverse array of microbes, including thermophilic and mesophilic fungi, cyanobacteria, marine and terrestrial actinobacteria, and pathogenic bacteria. Dependent on media conditions, on average and based on manual analysis, we observed 11.3 molecules associated with each microbial IMS experiment, which was split nearly 50:50 between secreted and colony-associated molecules. The spatial distributions of these metabolic exchange factors are related to the biological and ecological functions of the organisms. This work establishes that MALDI-based IMS can be used as a general tool to study a diverse array of microbes. Furthermore the article forwards the notion of the IMS platform as a window to discover previously unreported molecules by monitoring the metabolic exchange patterns of organisms when grown on agar substrates.

Root treatment with rhizobacteria antagonistic to Phytophthora blight affects anthracnose occurrence, ripening, and yield of pepper fruit in the plastic house and field.

We previously selected rhizobacterial strains CCR04, CCR80, GSE09, ISE13, and ISE14, which were antagonistic to Phytophthora blight of pepper. In this study, we investigated the effects of root treatment of rhizobacteria on anthracnose occurrence, ripening, and yield of pepper fruit in the plastic house and field in 2008 and 2009. We also examined the effects of volatiles produced by the strains on fruit ripening and on mycelial growth and spore development of Colletotrichum acutatum and Phytophthora capsici in the laboratory, identifying the volatile compounds by gas chromatography-mass spectrometry (GC-MS). In the house tests, all strains significantly (P < 0.05) reduced anthracnose incidence on pepper fruit; strains GSE09 and ISE14 consistently produced higher numbers of pepper fruit or increased the fresh weight of red fruit more than the controls in both years. In the field tests, all strains significantly (P < 0.05) reduced anthracnose occurrence on either green or red pepper fruit; strain ISE14 consistently produced higher numbers or increased fresh weights of red fruit more than the controls in both years. In the laboratory tests, volatiles produced by strains GSE09 and ISE13 only stimulated maturation of pepper fruit from green (unripe) to red (ripe) fruit; the volatiles of certain strains inhibited the growth and development of C. acutatum and P. capsici. On the other hand, GC-MS analysis of volatiles of strains GSE09 and ISE13 revealed 17 distinct compounds in both strains, including decane, dodecane, 1,3-di-tert-butylbenzene, tetradecane, 2,4-di-tert-butylphenol, and hexadecane. Among these compounds, 2,4-di-tert-butylphenol only stimulated fruit ripening and inhibited growth and development of the pathogens. Taken together, strains GSE09 and ISE14 effectively reduced anthracnose occurrence and stimulated pepper fruit ripening and yield, possibly via bacterial volatiles. Therefore, these two strains could be potential agents for controlling Phytophthora blight and anthracnose, and for increasing fruit ripening and yield. To our knowledge, this is the first report of volatiles such as 2,4-di-tert-butylphenol produced by rhizobacteria being related to both fruit ripening and pathogen inhibition.