Insecticidal features displayed by the beneficial rhizobacterium Pseudomonas chlororaphis PCL1606

Abstract

The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fluorescens group where insecticide fit genes have been found. Fit toxin, supported with other antimicrobial compounds, gives the bacterial capacity to repel and fight against predators, such as nematodes and insect larvae, thus protecting the plant host and itself. Pseudomonas chlororaphis PCL1606 is an antagonistic rhizobacterium isolated from avocado roots with efficient biocontrol against fungal soil-borne disease. The main antimicrobial compound produced by P. chlororaphis PCL606 is 2-hexyl,5-propyl resorcinol (HPR), which plays a crucial role in effective biocontrol against fungal pathogens. Further analysis of the P. chlororaphis PCL1606 genome showed the presence of hydrogen cyanide (HCN), pyrrolnitrin (PRN) and homologous fit genes. To test the insecticidal activity and to determine the bases for such activity, single and double mutants on the biosynthetic genes of these four compounds were tested in a Galleria mellonella larval model using inoculation by injection. The results revealed that fit toxin and HPR are the main compounds responsible for the insecticide phenotype in P. chlororaphis PCL1606, and HCN and PRN could be considered supporting compounds.

Introduction

The greatest variety of living beings is concentrated in the soil, and most of them gather around the roots of plants. Beneficial microorganisms coexist in this ecological niche along with pathogens and predators. Among the beneficial rhizobacteria associated with plant roots, the genus Pseudomonas is one of the most studied. The principal interactions of these rhizobacteria with the plants include cooperation with the plant host by plant growth promotion (PGPR), induction of systemic acquisition resistance (SAR; Haas and Défago 2005; Bakker et al. 2007), and competition or antagonism to soil-borne phytopathogens. In addition, these bacteria can show insecticidal activity and use insects as vectors for dispersal (Kupferschmied et al. 2013).

The main group of rhizobacteria of the genus Pseudomonas is the Pseudomonas fluorescens group, where insecticidal activity is not a common trait. Moreover, according to genomic diversity analysis, the P. fluorescens group is divided into three subclades (Loper et al. 2012). Interestingly, the strains belonging to subclade number 2 neither harbour the Fit toxin nor have the ability to kill Galleria mellonella larvae (Ruffner et al. 2015). However, all strains belonging to P. protegens and P. chlororaphis species tested in that study (which represent subclade 1) have both entomotoxicities (Ruffner et al. 2015). There are some examples of P. protegens and P. chlororaphis strains that are able to infect and efficiently kill insect larvae after oral uptake (Kupferschmied et al. 2013), and, this trait could also be related to the rhizobacterial ability to resist the deleterious effects of grazing predators, resulting in better host protection and competition against surrounding organisms (Nandi et al. 2015).

A close association with insecticidal activity has been demonstrated in the model bacterium P. protegens Pf-5 for a set of genes named the fitABCDEFGH cluster (fit, fluorescens insecticidal toxin; Péchy-Tarr et al. 2008, 2013; Ruffner et al. 2013). The unique virulence cassette harbours the fitD gene encoding the protein Fit toxin as well as regulatory and secretion protein functions (Péchy-Tarr et al. 2008). However, fitD deletion mutants retain substantial toxicity, indicating the presence of additional virulence factors (Péchy-Tarr et al. 2008; Ruffner et al. 2013). Many antagonistic pseudomonads are able to produce several antimicrobial compounds, such as phenazines (PHZ), 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), pyrrolnitrin (PRN), hydrogen cyanide (HCN), or cyclic lipopeptides, that form a cocktail able to repel plant pathogens (Haas and Keel 2003; Gross and Loper 2009; Rochat et al. 2010; Kupferschmied et al. 2013). It has been reported that some of these primary antifungal compounds can also be used for self-defence against predators, such as protozoa and nematodes (Bjørnlund et al. 2009; Jousset et al. 2009; Raaijmakers and Mazzola 2012). Specifically, HCN has shown nematicidal activity against Meloidogyne hapla, supported by PHZ and PRN production (Lee et al. 2011). Moreover, Nandi et al. (2015) reported that HCN and PRN were able to act as powerful fungal repellents; in addition, these two compounds are the key compounds that affect the interaction of P. chlororaphis PA23 and Caenorhabditis elegans. In this sense, Galleria mellonella larvae are being used as an insect model for antibiotic susceptibility testing (Tsai et al. 2016; Ignasiak and Maxwell 2017; Andrea et al. 2019), toxicity of chemicals (Allegra et al. 2018) and virulence factors (Durieux et al. 2021), revealing their usefulness for insecticidal activity studies of several rhizobacteria.

The model rhizobacterium P. chlororaphis PCL1606 was isolated from the root of healthy avocado trees (Persea Americana Mill.) in a crop infected by Rosellinia necatrix, the causal agent of white rot root. This bacterium is characterized by being a highly efficient antagonist against many soil-borne phytopathogenic fungi (Cazorla et al. 2006; Calderón et al. 2014). Analysis of antifungal compounds produced by PCL1606 has shown the production of HCN and PRN; however, its main antifungal antibiotic produced by PCL1606 is 2-hexyl,5-propyl resorcinol (HPR; Cazorla et al. 2006; Calderón et al. 2015). Previous studies using random insertional derivative Tn5 mutants and selected insertional defective mutants lacking HPR have indicated a direct correlation between resorcinol and the biocontrol of soil-borne fungal diseases (Cazorla et al. 2006; Calderón et al. 2013). Furthermore, the locus encoding a putative cytotoxin homologous to FitD has been allocated to the PCL1606 genome (Calderon et al. 2015). These circumstances suggest that P. chlororaphis PCL1606 could be a good candidate to have insecticidal functions.

The main goal of this study was to unravel the roles of HCN, PRN, HPR and FitD compounds in the insecticidal features of P. chlororaphis PCL1606. For this, the insecticidal activity of PCL1606 was tested in a Galleria mellonella (greater wax moth) model.

Materials And Methods

Strains Manipulation And Molecular Assays

Insertion mutagenesis for gene inactivation in P. chlororaphis PCL1606 was used to test the insecticide characteristics. Specifically, for this study, a disruptive vector was inserted into the putative fitD gene located on the PCL1606 chromosome via single-crossover homologous recombination (Arrebola et al. 2012). The cloning of vector pCR2.1 (Invitrogen Life Technologies USA) and plasmid purification were performed using standard procedures. The plasmids obtained were transformed into wild-type PCL1606 by electroporation (Choi et al. 2006). The double insertional mutant PCL1606::darBfitD was constructed using the previously obtained single insertional mutant PCL1606::darB (Calderón et al. 2013) in which the pCR2.1 derivative plasmid was already present in the mutant. Consequently, the suicide vector pJQ200SK (Table 1) was used to clone a fitD gene fragment and mutate the fitD gene by insertion of the chromosome via single-crossover homologous recombination.

The correct insertions of the disruption vectors were verified by PCR amplification. Bacterial growth curves were obtained in LB broth culture media to confirm similarities among the constructed defective mutants and the wild-type P. chlororaphis PCL1606 (data not shown).

Results

Genotypic and phenotypic study of the insecticidal capacity of P. chlororaphis PCL1606

An in silico analysis of putatively fit genes found in P. chlororaphis PCL1606 revealed a high similarity with fit gene operons detected in the insecticidal bacterium Pseudomonas protegens Pf-5, in which putative products are equivalents (Figure 1). Loci PCL1606_RS12165, PCL1606_RS12170 and PCL1606_RS12175 are related to the transport of cytotoxin, PCL1606_RS12180 is the largest locus and corresponds to the insect toxin FitD, PCL1606_12185 is annotated as an outer membrane protein, and PCL1606_RS12190, PCL1606_RS12195 and PCL1606_RS12200 are related to the regulation of toxin production. Equivalent genes were described in P. protegens Pf-5, where fitABC is related to transport by the type I secretion system, fitD has been described as a cytotoxin coding gene, fitE as a type I secretion outer membrane protein and fitFGH as a regulatory gene (Péchy-Tarr et al. 2008). Furthermore, the distribution and arrangement of these loci in the genome of PCL1606 are homologous to the fit cluster in P. protegens Pf-5. Likewise, the protein size and percentage of identity of the majority of loci with its equivalent fit gene showed that it was highly similar (Figure 1). Once the putative fitD gene was located in PCL1606, an insertional defective mutant was constructed to analyse its involvement in insecticide activity. Insecticidal activity using commercial Galleria mellonella larvae as a model by derivatising defective mutants of P. chlororaphis PCL1606 in antibiotic compounds, such as pirrolnitrine (PRN), hydrogen cyanide (HCN) and 2-hexyl,5-propyl resorcinol (HPR), was studied, as well as in the mutant defective in the global regulator GacS (Table 1). The results have been compared with the wild-type strains P. protegens Pf-5, where a fit cluster was described (Péchy-Tarr et al. 2008), P. chlororaphis subsp. aurantiaca BL915, a producer of HPR (Nowak-Thompson et al., 2003), and P. alcaligenes AVO110, whose biocontrol activity was not related to antibiotic production (Pliego et al. 2007; Pintado et al. 2021). Twenty-four hours postinoculation with 3x10⁵ cfu/mL as a dose (Figure 2), the control wild-type strains Pf-5, BL915 and PCL1606 caused a high insect mortality percentage (100% of dead larvae with intense larval melanization), contrary to AVO110, in which inoculation produced a comparable response to inoculation with the control buffer. Single mutants of P. chlororaphis PCL1606 in darB and fitD resulted in a slight decrease in mortality of approximately 15%, but they still had above 85% mortality in both cases. This drop in larval mortality shown by the PCL1606::darB mutant (defective in HPR) recovered to wild-type levels when this mutation was complemented (strain ComB). A decrease of approximately 40% in mortality occurred when single and double mutants in hcnB and prnC genes were tested. However, differences were observed when darB was involved in a double mutation with hcnB and prnC. Thus, in PCL1606 double mutants lacking HPR and PRN (PCL1606::darBprnC) or HPR and HCN (PCL1606::darBhcnB), the mortality levels observed increased to those shown by the wild-type strain values, which had higher mortality values than those displayed by the single mutant in darB, prnC or hcnB. A great difference was observed with the double mutant in the darB and fitD genes (PCL1606::darBfitD). The inoculation of this double mutant PCL1606::darBfitD decreased larval mortality to no inoculated control levels (Figure 2).

Involvement of HPR and Fit toxin in the toxicity of G. mellonella

A mortality survey eighty hours after inoculation with two different bacterial doses (3x10³ and 3x105 cfu/mL; Figure 3 and S1) was performed. The results showed that 40 h after inoculation, the wild-type P. chlororaphis PCL1606 and single mutants in darB, fitD and gacS retained the highest mortality; however, the double mutant PCL1606::darBfitD reduced the G. mellonella mortality level to the buffer control values. This double mutant reached 60% G. mellonella larval mortality 80 h after inoculation. Therefore, there was a delay in the insecticidal activity of PCL1606 when HPR and Fit toxin were impaired in the same strain (Figure 3a). With the higher dose, all events reached the highest mortality from 16 h to 30 h after inoculation, but the double mutant HPR-Fit toxin showed a delay in killing the larvae, reaching 100% mortality 60 h after inoculation (Figure S1).

The symptoms observed in G. mellonella larvae 24 h after the injection (3x10³ cfu/mL) with fitD- and darB-defective mutants (Figure 3b) showed that several larvae started to melanise. After 30 h, nearly all the larvae inoculated with the single mutants PCL1606::fitD, PCL1606::darB and PCL1606::gacS and the wild-type PCL1606 showed dark melanization. However, at 30 h postinoculation with buffer or with double mutant PCL1606::darBfitD, the inoculated larvae did not display signs of melanization and mobility, and only 60 h after inoculation, they began to show symptoms of larval intoxication in those infected with the HPR-Fit toxin double mutant (Figure 3b). The different mortality kinetics could be better distinguished at 30, 40 and 60 h after inoculation (Figure S2), showing that the wild-type PCL1606 strain presented progressively increasing mortality, reaching 100% at 60 h. The single mutants in fitD and darB showed significantly higher virulence than the wild-type strain at 30 h. The single mutant in gacS seemed to be delayed until 40 h to display higher virulence. Finally, the double mutant seemed to be innocuous after 60 h, when insecticide activity began (Figure S2).

Discussion

Pseudomonas chlororaphis PCL1606 is a rhizobacterum isolated from avocado roots characterized by 2-hexyl,5-propyl resorcinol (HPR) production, the main compound involved in biocontrol (Cazorla et al. 2006), but also in other relevant phenotypes, such as biofilm formation and colonization (Calderón et al. 2019; Arrebola et al. 2019). A previous analysis of additional antifungal antibiotic compounds produced by PCL1606 revealed the production and presence of coding genes for hydrogen cyanide (HCN) and pyrrolnitrin (PRN); however, phenazine production and the presence of coding genes were not found in PCL1606 (Calderón et al. 2015). Additionally, the presence of genes homologous to the fitABCDEFGH cluster, encoding a putative cytotoxin similar to Fit toxin, was detected in the genome of P. chlororaphis PCL1606 (Calderón et al. 2015). According to Ruffner et al. (2015), the Fit cytotoxin is restricted to a particular group of rhizobacteria comprised of P. protegens and P. chlororaphis, and it is strongly correlated with high insect toxicity.

PCL1606 displayed insecticidal activity at the same level as the control strains P. chlororaphis subsp aurantiaca (former P. aurantiaca) BL915 and P. protegens Pf-5, where HPR and Fit production were first described, respectively (Nowak-Thompson et al. 2003; Péchy-Tarr et al. 2008). On the other hand, the nonantagonistic rhizobacterium P. alcaligenes AVO110 did not show insecticidal capacity, did not have the fit or any antifungal antibiotic genes in its genome (Pintado et al. 2021), and did not produce antifungal secondary metabolites (Pliego et al. 2007; Pintado et al. 2021). The typical symptoms produced by the wild-type strain PCL1606 correspond to mortality accompanied by intense melanization of the G. mellonella larvae from 24 h postinoculation. Melanization is a typical insecticidal symptom corresponding to the synthesis and deposition of melanin to encapsulate pathogens at the wound site followed by haemolymph coagulation and opsonisation (Tsai et al. 2016).

Single and double mutants impaired the production of antifungal hydrogen cyanide (HCN) or pyrrolnitrin (PRN) compounds in PCL1606, resulting in a mortality reduction of G. mellonella larvae, but with no differences among them. This suggests that both compounds could only contribute to the insecticidal background and were not the main insecticidal compounds produced, as previously described for other insecticidal strains (Flury et al. 2017). It has been previously described that the insecticidal virulence of P. protegens CHA0 and P. chlororaphis PCL1391 mutants lacking one or several antibiotics, such as 2,4-diacetylphloroglucinol, phenazine, pyrrolnitrin or pyoluterin, was also not reduced (Flury et al. 2017).

The role of HCN and PRN on insecticidal and nematicidal activity by antagonistic bacteria has been illustrated by other authors (Nandi et al. 2015; Kang et al. 2019), and our results indicate their involvement in the insecticidal phenotype displayed by the model strain PCL1606. In addition, this study included the PCL1606 derivative mutant defective in gacS, since gacS is a global regulator that can interfere with activities related to the production of secondary compounds, such as antifungals and insecticidals (Saraf et al. 2011; Nandi et al. 2015). Previous studies have reported that mutants deficient in the global regulator gacS showed no insecticide activity (Kang et al. 2019). These results do not agree with our observation where the impaired mutants in the gacS gene of PCL1606 still retain insecticidal activity, suggesting a gacS-regulated independent pathway responsible for the insecticidal phenotype.

For this, the effect of HPR and Fit products was studied. Single mutants of these genes scarcely lowered G. mellonella larval mortality, but the absence of both genes completely impaired the insecticidal activity of the derivative bacteria, even in the presence of hcnB and prnC genes.

The insecticidal and nematicidal effectiveness of the Fit cytotoxin in P. protegens and P. chlororaphis has been documented (Kupferschmied et al. 2013; Péchy-Tarr et al. 2008). Heterologous expression of the Fit toxin in Escherichia coli resulted in the capacity of the bacterium to kill the insect host upon injection (Kupferschmied et al. 2013). In addition, the deletionally defective fitD mutants of P. protegens Pf-5 and CHA0 show a decrease in insecticidal activity, demonstrating that FitD makes an important contribution to insect virulence but may not be essential for the capacity for these bacteria to multiply in the infected host. The fact that the fitD mutants still retained a certain level of toxicity indicated to the authors that additional factors may contribute to anti-insect activity (Péchy-Tarr et al. 2008), in agreement with our observations on PCL1606. However, the PCL1606 derivative mutant with the fit toxin gene disrupted showed the capacity to kill G. mellanella at almost 80% 30 h postinfection. This result is not only due to HCN and PRN, as reported by the double derivative mutant in HPR and Fit toxin, with no virulence at this time postinfection. On the other hand, the double mutant HCN and PRN still showed that 60% of G. mellonella larvae died. Therefore, we can conclude that HCN and PRN could contribute to the insecticidal capacity of PCL1606, but they could be considered accompanying compounds. Thus, HPR and the Fit toxin could be considered the main toxins responsible for insecticidal activity. Single derivative mutants in HPR and Fit toxin displayed the same levels of G. mellonella mortality, suggesting that the mutation of one of them can complement the mortality index by the other gene and vice versa. However, the mortality shown by the derivative mutant PCL1606::darBfitD was innocuous after approximately one day and a half, when the first sign of mortality started to appear.

HPR has been described as the main antibiotic against fungal pathogens such as Rosellinia necatrix or Fusarium oxysporum (Cazorla et al. 2006). However, HPR has been revealed as a versatile compound involved in bacterial adhesion, colony morphology and typical air-liquid interphase pellicles produced by PCL1606 (Calderón et al. 2019). Due to the chemical nature of HPR, it is possibly not an integral component of the biofilm matrix, together with the evidence of a regulatory role in Photorhabdus asymbiotica (Hapeshi and Waterfield 2017), which extends its putative role in PCL1606.

The combined antimicrobial and regulatory roles of HPR in PCL1606 could explain several results obtained in the present study. The toxic nature of HPR against different organisms (Kanda et al. 1975) could directly affect G. mellonella cells, as happens with antifungal phenazines (Wand et al. 2013), helping the insecticidal characteristics of P. chlororaphis PCL1606. This insecticidal activity would rival FitD in toxicity, thus justifying the single mutants PCL1606:darB and PCL1606:fitD results in comparison with double mutant PCL1606::darBfitD, also revealing that because of the absence of HPR and Fit toxin, there was no mortality due to HCN and PRN. On the other hand, HPR could have additional regulatory roles on secondary metabolites since alkylresorcinols can be involved as signalling molecules in a novel quorum sensing two-component regulatory system (Brameyer et al. 2015). Belonging to these sets of secondary metabolites could be chitinase and phospholipase C, putative genes that have been found in the PCL1606 genome (PCL1606_RS13585 as chitinase and PCL1606_RS14060 as phosphatase C). Both enzymes have also been reported to be important in insect pathogenicity (Flury et al. 2016). This also helps to explain the gacS mutant results, whose killing ability was not affected, suggesting a regulation of all the compounds at a higher hierarchy, but this regulatory role of HPR is still a hypothesis under study.

In summary, PCL1606 could produce the Fit toxin, a described compound with insecticidal capacity, and HPR, which has been shown to have insecticidal potential, to which its fungicidal character and its possible regulatory role must be added. This confirms HPR as one the main compounds produced by P. chlororaphis PCL1606 involved in the beneficial phenotypes displayed by this model bacterium.

Declarations

Acknowledgements

Thank to Mrs. Irene Linares Rueda for their technical assistance with laboratory research.

Data and materials availability 

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request

Funding

This work was supported by research project AGL2017-83368-CO2-1-R of Ministerio de Ciencias y Tecnología, and the project UMA-FEDERJA-046 of Junta de Andalucía. 

Competing Interests

The authors declare they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author’s contribution

Eva Arrebola: Conceptualization, Data curation, Investigation, Formal analysis, Writing original draft. Francesca R. Aprile and Claudia E. Calderón: Assays performant and results. Antonio de Vicente: Conceptualization and writing support. Francisco M. Cazorla: Funding acquisition, Methodology, Supervision, Writing, review and editing. 

Ethics approval: This article does not contain any studies with human or animal subjects performed by any of the authors

Consent to participate: No applicable

Consent for publication: No applicable

Conflict of interest: The authors declare no competing interests

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