细菌群体感应抑制剂研究进展

多重耐药菌的出现已成为农业防治植物病害的一大难题,目前急需发展新的防治策略。群体感应 (quorum sensing, QS) 是一种微生物之间普遍存在的依赖于菌体密度的沟通协调机制,控制着细菌的生长、增殖、致病性、生物被膜形成及相关群体活动行为。群体感应抑制剂在抑制细菌毒性基因表达时不会对细菌生长产生压力,从而避免了细菌耐药性的产生。这一新颖的抑菌机制使其在开发新型农药方面有很大潜力。本文着重介绍了细菌群体感应机制、天然的及合成的细菌群体感应抑制剂种类及其应用。     

Identification of <Emphasis Type="Italic">N</Emphasis> −3-hydroxyoctanoyl-homoserine lactone production in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> 5064, pathogenic to broccoli,and controlling biosurfactant production by quorum sensing

Quorum sensing controls a number of key processes in growth, reproduction and virulence of many gram-negative bacteria via signalling molecules or autoinducers. It can control, for instance, the production of pectic enzymes which are virulence factors in disease. Pseudomonas fluorescens 5064 produces biosurfactants which are important for bacterial establishment on the plant surface prior to causing disease in broccoli. The aim of this paper was to determine if biosurfactant production in this bacterium is controlled via quorum sensing. To do this, 35 surfactant-minus Tn5 mutants of P. fluorescens 5064 were screened for their abilities to produce a quorum sensing signal. Six of these biosurfactant-deficient mutants showed a large reduction in quorum sensing signal production and varied in their abilities to cause disease. In one mutant, 6423, which contains a single Tn5 insertion, the production of the signal was almost eliminated. Synthetic quorum sensing signal and quorum sensing signal extracted from wild type P. fluorescens 5064 restored biosurfactant production by addition to the culture in mutant 6423. The quorum sensing signal in wild type P. fluorescens 5064 was identified by high pressure liquid chromatography and mass spectrometry as N -3-hydroxyoctanoyl-homoserine lactone.     

Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a disease that affects almost all types of citrus crops. Production of particular Xcc pathogenicity factors is controlled by a gene cluster rpf, which encodes elements of a cell–cell communication system called quorum sensing (QS), mediated by molecules of the diffusible signal factor (DSF) family. Interference with cell–cell signalling, also termed quorum quenching, either by signal degradation or over‐production, has been suggested as a strategy to control bacterial disease. In this study, three bacterial strains were isolated from citrus leaves that displayed the ability to disrupt QS signalling in Xcc. Pathogenicity assays in sweet orange (Citrus sinensis) showed that bacteria of the genera Pseudomonas and Bacillus also have a strong ability to reduce the severity of citrus canker disease. These effects were associated with alteration in bacterial attachment and biofilm formation, factors that are known to contribute to Xcc virulence. These quorum‐quenching bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used to treat citrus canker disease.     

Genetic and physiological evidence for the production of N-acyl homoserine lactones by Pseudomonas syringae pv. syringae and other fluorescent plant pathogenic Pseudomonas species

N-acyl homoserine lactones (AHLs) function as cell density (quorum) sensing signals and regulate diverse metabolic processes in several gram negative bacteria. We report that strains of Pseudomonas syringae pvs. syringae (Pss), tabaci and tomato as well as P. corrugata and P. savastanoi produce difussible AHLs that activate the lux operons of Vibrio fischeri or the tra::lacZ fusion of Agrobacterium tumefaciens. In Pss strain B3A, AHL production occurs in cell density dependent manner. Nucleotide sequence and genetic complementation data revealed the presence of ahlI_Pss, a luxI homolog within the Ahl⁺ DNA of Pss strain B3A. The DNA expresses in AHL-deficient strains of P. fluorescens and E. carotovora subsp. carotovora (Ecc), and restores extracellular enzyme production and pathogenicity in the Ecc strain. The derivatives of Pss strains B3A and 301D carrying chromosomal ahlI::lacZ do not produce AHL, but like their wild type parents, produce extracellular protease and the phytotoxin syringomycin as well as elicit the hypersensitive reaction in tobacco leaves. While these strains also produce a basal level of -galactosidase activity, the expression of ahlI::lacZ is substantially stimulated in the presence of multiple copies of the DNA or by the addition of cell-free spent cultures containing AHL. The activation of -galactosidase production occurs with spent cultures of some, but not all Pseudomonas strains which produce AHL as indicated by the Lux and tra::lacZ assays. Pss strains deficient in the global regulatory genes, gacA or lemA, produce very low levels of AHL. Since inactivation of ahlI_Pss eliminates AHL production and since Ahl⁺ Pseudomonas strains carry the homolog of ahlI_Pss, we conclude that ahlI_Pss specifies a key step in AHL biosynthesis and it has been conserved in many plant pathogenic pseudomonads.     

Plant responses to plant growth-promoting rhizobacteria

Non-pathogenic soilborne microorganisms can promote plant growth, as well as suppress diseases. Plant growth promotion is taken to result from improved nutrient acquisition or hormonal stimulation. Disease suppression can occur through microbial antagonism or induction of resistance in the plant. Several rhizobacterial strains have been shown to act as plant growth-promoting bacteria through both stimulation of growth and induced systemic resistance (ISR), but it is not clear in how far both mechanisms are connected. Induced resistance is manifested as a reduction of the number of diseased plants or in disease severity upon subsequent infection by a pathogen. Such reduced disease susceptibility can be local or systemic, result from developmental or environmental factors and depend on multiple mechanisms. The spectrum of diseases to which PGPR-elicited ISR confers enhanced resistance overlaps partly with that of pathogen-induced systemic acquired resistance (SAR). Both ISR and SAR represent a state of enhanced basal resistance of the plant that depends on the signalling compounds jasmonic acid and salicylic acid, respectively, and pathogens are differentially sensitive to the resistances activated by each of these signalling pathways. Root-colonizing Pseudomonas bacteria have been shown to alter plant gene expression in roots and leaves to different extents, indicative of recognition of one or more bacterial determinants by specific plant receptors. Conversely, plants can alter root exudation and secrete compounds that interfere with quorum sensing (QS) regulation in the bacteria. Such two-way signalling resembles the interaction of root-nodulating Rhizobia with legumes and between mycorrhizal fungi and roots of the majority of plant species. Although ISR-eliciting rhizobacteria can induce typical early defence-related responses in cell suspensions, in plants they do not necessarily activate defence-related gene expression. Instead, they appear to act through priming of effective resistance mechanisms, as reflected by earlier and stronger defence reactions once infection occurs.     

Silencing of Erwinia amylovora sy69 AHL‐quorum sensing by a Bacillus simplex AHL‐inducible aiiA gene encoding a zinc‐dependent N‐acyl‐homoserine lactonase

Quorum sensing in Gram‐negative bacteria is regulated by diffusible signal molecules called N‐acyl‐l ‐homoserine lactones (AHLs). These molecules are degraded by lactonases. In this study, six Bacillus simplex isolates were characterized and identified as a new quorum‐quenching species of Bacillus. An aiiA gene encoding an AHL‐lactonase was identified based on evidence that: (i) it showed high homology with other aiiA genes of Bacillus sp.; (ii) the deduced amino acid sequence contained two conserved regions, ¹⁰⁴SHLHFDH¹¹¹ and ¹⁶⁵TPGHTPGH¹⁷³, characteristic of the metallo‐β‐lactamase superfamily; and (iii) the protein had zinc‐dependent AHL‐degrading activity. Additionally, the expression of the aiiA gene was significantly up‐regulated by 3‐oxo‐AHL. The AHL‐lactonase inhibited multiplication of the 3‐oxo‐C6‐AHL‐producing plant pathogen Erwinia amylovora sy69 both in vitro and in planta. The results provide support for the use of the quorum‐quenching functionality of B. simplex in the integrated control of the devastating fire blight pathogen.     

Roles of diffusible signals in communication among plant-associated bacteria

ABSTRACT In nature, Pseudomonas species compete and co-exist in mixed communities with a diversity of prokaryotic and eukaryotic micro- and macroorganisms. Many bacteria produce various signals that control gene expression and thus contribute to specific bacterial behaviors and coordinate essential functions with other members of the community. The best-studied signaling compounds are N-acyl-homoserine lactones (AHLs), which are involved in quorum sensing (QS) regulation and are produced by a diverse range of bacterial taxa. To date, research on QS has focused on how signals control gene expression in the bacterial cell and the role of these signals in positive and negative communication among different groups of organisms. Additionally, mechanisms for AHL decay and AHL utilization as sole carbon/energy sources have been identified. Some host organisms produce compounds that can mimic AHLs, and some bacterial signals can influence host gene expression. Thus, interkingdom communication may be more widespread than previously believed. Our current understanding of individual, community and bacterial-host interactions is still in its infancy and there are many exciting discoveries yet to be made.     

Bacterial Lipopolysaccharides And Plant—pathogen Interactions

Lipopolysaccharides are amphipathic molecules forming the outermost layer of the cell surface of Gram-negative bacteria. They are essential for protecting the cell from hostile environments and, in the case of pathogens, they play a direct role in interactions with eukaryotic host cells. Mutants with altered lipopolysaccharide structure have been obtained with several plant pathogenic bacteria; such mutants generally show reduced virulence. Purified lipopolysaccharide has several effects on plants, notably suppression of the hypersensitive response to subsequently inoculated avirulent pathogens. The suppression is strictly localized and is observed within a time window of, typically, 10–30 h. Although infiltration of lipopolysaccharide into leaves produces no macroscopic symptoms, characteristic changes in plant gene expression can be observed. One effect is to sensitize the plant tissue to subsequent bacterial inoculation so that the sensitized tissue responds more rapidly and intensely, giving partial inhibition of bacterial growth. The synthesis of antimicrobial hydroxycinnamoyl tyramine conjugates is one facet of the process which provides an excellent biochemical model for analysing the phenomenon. Lipopolysaccharide induces the synthesis of two enzymes involved in conjugate production (tyrosine decarboxylase and tyramine-hydroxycinnamoyl transferase), but the conjugates themselves are not produced until bacteria are subsequently inoculated. Using this and other examples we discuss the mechanisms of lipopolysaccharide action on plants in the context of plant disease.     

Soil applications of acibenzolar-<Emphasis Type="Italic">S</Emphasis>-methyl induce defense gene expression in tomato plants against bacterial spot

Acibenzolar-S-methyl (ASM), a plant activator known to induce plant resistance, has been used as foliar sprays to manage several plant diseases including bacterial spot on tomato caused by four distinct Xanthomonas species. This study aimed to investigate the effects of soil application rates of ASM on bacterial spot of tomato and the expression levels of the two pathogenesis-related (PR) genes, PR1a and PR1b, in leaf tissues. Tomato seedlings were leaf-applied with ASM at 18.8 mg/l corresponding to the labeled rate, soil-applied with ASM at 0.84 and 10 mg/l, and sprayed with water served as an untreated control. The soil application of ASM at 10 mg/l consistently reduced the final disease severity and disease progress compared to the untreated control in four growth chamber experiments, whereas the soil application of ASM at 0.84 mg/l and foliar spray of ASM significantly reduced the final disease severity and area under disease progress curve (AUDPC) in three out of the four experiments. The expression levels of PR1a and PR1b in the leaf tissues were significantly induced by both soil and foliar applications of ASM. In addition, field trial results suggested that the soil applications of ASM at 10 mg/l markedly reduced disease progress compared to the control and copper standard. Although the control efficiency of soil applications of ASM depends on rates used, this study suggests that ASM can be used as soil applications to induce tomato resistance against bacterial spot.     

酰基高丝氨酸内酯酶SS10的酶学特性及其抗软腐病功能的初探

 N-酰基高丝氨酸内酯(AHLs)作为细菌群体感应中的信号分子参与调节植物病原细菌致病因子的表达。N-酰基高丝氨酸内酯酶(简称AHL内酯酶)通过水解AHL生成酰基高丝氨酸, 使AHL失去活性, 阻断病原菌的群体感应机制, 使病原菌失去致病力。利用谷胱甘肽-琼脂糖亲和层析柱和凝血酶处理获得纯化的重组AHL内酯酶SS10, 分子量约为28 kD, 其反应的最适pH值为8.0, 在30℃以下稳定性很高。动力学和底物特异性分析表明:AHL内酯酶SS10对所检测的8种AHL具有很强的催化活性, 表明该酶的底物谱可能较宽, 并且具有催化裂解酰基高丝氨酸内酯键的特异性。纯化的AHL内酯酶SS10可以显著降低胡萝卜软腐欧文氏菌胞外果胶酶、多聚半乳糖醛酸酶的产生量。致病性测定表明, 该重组蛋白对胡萝卜软腐病菌具有较强的抗病活性。     

Conserved virulence factor regulation and secretion systems in bacterial pathogens of plants and animals

Recent research has revealed the emergence of common themes in the molecular mechanisms of virulence in bacterial pathogens of plants and animals. In particular, the systems used for the global control of virulence factor synthesis and for the secretion of virulence determinants in diverse bacterial pathogens show strong conservation, implying evolutionary relatedness. Global control of virulence factor synthesis can be affected by a variety of environmental factors, bacterial hormones and programmed genetic rearrangements.Protein secretion in Gram-negative bacteria occurs via a number of targeting pathways. Type I and type III secretion systems mediate translocation across both the inner and outer membrane in a single step, while type II secretion proceeds via a periplasmic intermediate. Type II and type III secretion systems have been shown to target virulence determinants in both plant and animal bacterial pathogens.Abbreviations Cel cellulase - EPS extracellular polysaccharide - HL homoserine lactone - OHHL N-(3-oxohexanoyl)-l-homoserine lactone - PC phenotypic conversion - Pel pectate lyase - pv. pathovar - spp. species     

The critical role of biofilms in bacterial vascular plant pathogenesis

Bacterial life is a combination of two lifestyles, mobile and social. In the social lifestyle, cells are usually embedded in a self-produced matrix and attached to biotic or abiotic surfaces. These communities can be organized as either single or multilayered structures termed biofilms. Biofilms evolved to cope with the harsh environmental conditions that bacteria encounter within the host, mostly from the host’s defence response. In plant pathogenic bacteria, biofilms participate in the whole process of pathogenicity, from the first step of invasion to the full colonization of plant tissues. The specific role that biofilms play in the pathogenicity process of plant bacterial pathogens is poorly understood. In this review, the role of biofilms in the pathogenic process of major vascular plant pathogens is examined. In addition, quorum sensing signals and components that are essential for biofilm formation and therefore, for pathogenesis, are addressed. Although, in certain systems, further research is required, experimental evidence in the literature indicates that biofilms are, in most cases, essential for pathogenesis.     

水稻病原菌Burkholderia plantarii群体感应和群体淬灭研究进展

植物伯克霍尔德菌Burkholderia plantarii是引起水稻秧苗细菌性立枯病的重要病原菌之一,其侵染性、繁殖力及适应性均很强,严重威胁中国水稻生产。文章围绕B.plantarii的发生、危害及致病机理,着重论述了细菌群体感应系统(quorum sensing,QS)的生理功能及其在B.plantarii致病力调控方面的最新研究进展,并进一步从根际微生物互作角度,综述了种间信号分子对病原菌群体淬灭(quorum quenching)的作用机制,同时结合种间信号分子的独特性,展望了其在新型微生物杀菌剂研发中的重要性和应用潜力。     

Efficacy of some rhizospheric and endophytic bacteria <Emphasis Type="Italic">in vitro</Emphasis> and as seed coating for the control of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> infecting durum wheat in Tunisia

Sixty two rhizospheric and endophytic bacterial strains were evaluated for their biocontrol effect on two aggressive Fusarium culmorum isolates (Fc2 and Fc3). We observed that 35 % and 23 % of the tested strains inhibited the in vitro growth of Fc2 and Fc3 respectively. The observed antagonism was due to inhibition by contact (13–19 % of the strains) or at distance (10–16 % of the strains) for both fungal isolates. Some of the antagonistic bacteria showed the ability to produce diffuse and/or volatile compounds that inhibit the growth, the sporulation and macroconidia germination of F. culmorum. None of the tested antagonistic bacteria showed chitinase activity on synthetic medium. The sequencing of the 16S rDNA genes of some antagonistic bacteria showed that they belong to the genera Bacillus, Pseudomonas and Microbacterium. The double inoculation of durum wheat seeds by the antagonistic bacterial strains (B13, B18, BSE1, BSE3 and B16E) and the two F. culmorum isolates showed that germination and seedling vigor were generally improved in vitro. The percentage of infected seeds was also reduced. In greenhouse trials, the biocontrol effectiveness of F. culmorum was dependant from the virulence of the fungal strain and the specificity of the antagonistic interaction between bacterial and fungal strains. The bacterial strains B18 and B16E reduced F. culmorum infection on durum wheat plants probably due to their antagonistic and plant growth promoting activities and they may be used in a mixture as seed biopriming inoculum for plant growth bio-promoting and Fusarium wheat diseases biocontrol.     

Pseudomonas for biological control of dutch elm disease. I. Labeling,detection and identification of pseudomonas isolates injected into elms; comparison of various methods

To understand the mechanisms involved in biological control of Dutch elm disease byPseudomonas, data were needed on the distribution of the introduced bacteria within elm and on the development of the bacterial population over a period of time.As traditional biochemical identification techniques are not suitable for distinguishment between individualPseudomonas isolates, three alternative approaches were compared.

Induction of systemic resistance,root colonisation and biocontrol activities of the rhizospheric strain of <Emphasis Type="Italic">Serratia plymuthica</Emphasis> are dependent on N-acyl homoserine lactones

Quorum sensing regulation, mediated by N-acyl homoserine lactone signals, produced by strain Serratia plymuthica HRO-C48 isolated from the rhizosphere of oilseed rape, was found to be responsible for this strain’s ability to produce the broad spectrum antibiotic pyrrolnitrin. In this study, we have shown that some other biocontrol-related traits of strain HRO-C48, such as protection of cucumbers against Pythium apahnidermatum damping-off disease, induced systemic resistance to Botrytis cinerea grey mold in bean and tomato plants, and that colonisation of the rhizosphere also depends on AHL signalling. The results prove that quorum sensing regulation may be generally involved in interactions between plant-associated bacteria, fungal pathogens and host plants.     

Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid and accurate identification of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> and <Emphasis Type="Italic">Ralstonia pseudosolanacearum</Emphasis>

Ralstonia solanacearum “species complex” (RSSC) represents soil-borne plant pathogenic bacteria, consisting of diverse and widespread strains that cause bacterial wilt on a wide range of host plants. A recent polyphasic taxonomic study has divided the RSSC into three bacterial species; Ralstonia pseudosolanacearum (phylotypes I and III), Ralstonia solanacearum (phylotype II) and Ralstonia syzygii (phylotype IV). Currently, standard identification of RSSC in plant health laboratories mainly relies on performance of two tests that are based on a different principle. However, these tests are inadequate to precisely discriminate among the three bacterial species in the RSSC. The accurate identification of each of the three bacterial species in the RSSC requires additional molecular tests, including a phylotype determination. These methodologies are labor-intensive, time consuming and rather impractical for routine identification purposes in a plant health laboratory. We explored the potential for an accurate identification of R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II) in RSSC, upon implementation of the MALDI-TOF MS tool, and after the creation and validation of an in-house database supplementing the commercial database and covering the entire known genetic diversity in RSSC. MALDI-TOF MS is an emerging approach for identification of bacterial plant pathogens and has been shown to be robust and reproducible. Additionally, when compared to the conventional microbial identification methods it is shown to be less laborious and less expensive. Validation data demonstrated that our in-house database (Mass Spectra Profiles, MSPs) was very specific resulting in the rapid and accurate identification of Ralstonia solanacearum (phylotype II), and Ralstonia pseudosolanacearum (phylotypes I and III). Additionally, no false positive results were obtained with our in-house database for other related Ralstonia sp., such as the R. picketii isolate PD 3286, or for the Pseudomonas syringae and Pseudomonas spp. isolates.     

群体感应信号物质对吡咯伯克霍尔德氏菌JK-SH007在杨树内定殖的影响

为了明确杨树溃疡病生防菌吡咯伯克霍尔德氏菌JK-SH007的群体感应系统是否与其内生定殖相关，本文通过群体感应指示菌紫色杆菌CV026和液相色谱串联四级杆飞行时间质谱技术（HPLC-Q-TOF-MS）确定其群体感应信号物质种类，并利用结晶紫染色、菌体回收、GFP标记等技术，研究其群体感应信号物质对该菌株生物膜及在杨树苗内的定殖能力的影响。结果表明，菌株JK-SH007产生的群体感应信号物质为含8个碳原子酰基侧链的辛酰基-L-高丝氨酸内酯（C₈-HSL）。群体感应信号物质C₈-HSL的添加对该菌株生物膜及在杨树苗内的定殖能力有影响，并且呈现低浓度促进，较高浓度抑制的规律。C₈-HSL的添加与菌株JK-SH007生物膜的OD值和菌体量具有显著相关性，相关系数分别为0.923和0.756。当添加1.0% C₈-HSL终浓度达到5×10^{-8 g/L}时，菌株JK-SH007生物膜的形成量达到峰值。荧光显微镜镜检发现，当添加100 μL C₈-HSL时，杨树组培苗根和茎中GFP荧光标记的菌株JK-SH007的定殖数量明显较CK多。菌株JK-SH007的群体感应与其在杨树内的内生定殖能力密切相关，群体感应信号物质C₈-HSL具有增强菌株JK-SH007内生定殖的能力，同时也表明该物质有利于该菌株生防效果的发挥。     

Quorum sensing as a target for developing control strategies for the plant pathogen <Emphasis Type="Italic">Pectobacterium</Emphasis>

Quorum sensing is a regulatory mechanism that connects gene expression to cell density in bacteria. Amongst proteobacteria, numerous functions are regulated in this way, including pathogenicity in the Enterobacteriaceae genus Pectobacterium. In Pectobacterium, the signalling molecules involved in this regulatory process belong to the N-acyl-homoserine lactone class. Over the last 6 years, various studies have shown that these signal molecules could be degraded by other bacteria or by plant and animal cells, opening the path to innovative biocontrol strategies. This review explores the various determinants of pathogenicity in Pectobacterium and describes approaches that have been developed to quench the quorum-sensing-dependent pathogenicity in Pectobacterium. These approaches range from signal degradation by physicochemical constraints to the identification of signal-sensing inhibitors and from the identification of enzymes degrading acyl-homoserine lactones to the construction of transgenic plants tolerant to Pectobacterium.     

Relative potency of culture supernatants of <Emphasis Type="Italic">Xenorhabdus</Emphasis> and <Emphasis Type="Italic">Photorhabdus</Emphasis> spp. on growth of some fungal phytopathogens

In previous research, concentrated metabolites produced by bacteria of the genera Xenorhabdus and Photorhabdus (which are symbionts of entomopathogenic nematodes) were reported to be highly suppressive to fungal and oomycete plant pathogens. Conceivably, application of non-concentrated bacterial filtrates would be more economically feasible compared to using concentrated metabolites. We evaluated the potency of 10 % v/v cell-free supernatants of the bacteria X. bovienii, X. nematophila, X. cabanillasii, X. szentirmaii, P. temperata, P. luminescens (VS) and P. luminescens (K22) against Fusicladium carpophilum (peach scab), F. effusum (pecan scab), Monilinia fructicola (brown rot), Glomerella cingulata (anthracnose) and Armillaria tabescens (root rot). A bioactive compound derived from Photorhabdus bacteria, trans-cinnamic acid (TCA), was also compared with the bacterial filtrates. Fungal colony size based on manual measurements was compared for accuracy to measurements taken by image analysis. Supernatants of Xenorhabdus spp. exhibited stronger suppressive effects on spore germination and vegetative growth when compared with Photorhabdus spp. Overall, TCA was the most effective treatment; vegetative growth was completely inhibited by TCA (1.27 mg/ml). TCA treatments also suppressed spore germination of F. carpophylium and F. effussum by approximately 90 %. The efficacy of supernatants varied among Xenorhabdus species depending on the species tested, but X. szentirmaii filtrates tended to cause greater inhibition relative to the other bacteria supernatants. Manual measurement of colony diameter required at least two replicate estimates of the colony to avoid a type II error. Area measurements were slightly overestimated based on ruler measurements, but did not affect the outcome of the analysis. Supernatants of Xenorhabdus spp., Photorhabdus spp., or TCA, did not cause any phytotoxic effects when applied to various plant species in the greenhouse. Our results indicate the potential of using TCA or Xenorhabdus cell free supernatants as bio-fungicides. Such a product, based on bacterial culture supernatants, would be economically viable, marketable and easily applicable by the end-users in many situations.