Two Philippine Photorhabdus luminescens strains inhibit the in vitro growth of Lasiodiplodia theobromae, Fusarium oxysporum f. sp. lycopersici, and Colletotrichum spp.

Abstract

Background

Fungal phytopathogens are one of the leading causes of loss in global food production. Chemical fungicides have always been used to control the phytopathogens to mitigate losses. However, it is widely known that this approach is not sustainable. Thus, it is essential to develop alternative control methods, such as the use of biological control agents.

Results

This study provided a preliminary data on the efficacy of 2 local Photorhabdus strains, associated with Heterorhabditis indica BSDS and H. indica MAP, against selected post-harvest fungal phytopathogens, Fusarium oxysporum f. sp. lycopersici, Lasiodiplodia theobromae, Colletotrichum musae, and another Colletotrichum sp., by measuring their in vitro inhibitory activity. The Photorhabdus strains were isolated from the hemolymph of Ostrinia furnicalis infected with H. indica BSDS and H. indica MAP and grown selectively on NBTA media. Firstly, the bacterial endosymbionts' generic identity was confirmed through colony PCR based on a Photorhabdus Txp40 toxin-specific marker. Species identity was then elucidated through 16s marker-assisted GenBank mining as P. luminescens, sharing 99.51–99.58% similarity with P. luminescens subsp. akhurstii (Accession no. AY278643.1). Anti-fungal activity was observed by the bioassay experiments, using cell-free culture filtrates (CFCs), obtained from P. luminescens tryptic soy broth suspensions (OD₆₀₀ = 2.0) amended in PDA medium (25%v/v) based on percentage growth inhibition. The CFCs of P. luminescens BSDS showed a significantly higher suppressive activity against Colletotrichum musae, Colletotrichum sp., and Lasiodiplodia theobromae, with 93.18 ± 0.46%, 74.15 ± 0.54%, and 60.51 ± 2.04% growth inhibition, respectively, while the CFC of P. luminescens MAP showed a significantly higher suppressive activity against F. oxysporum f. sp. lycopersici with 21.87 ± 0.71% growth inhibition.

Conclusions

The results strongly showed that these strains of Photorhabdus can be promising biological control agents against these fungal phytopathogens. Further extensive research is warranted for the development of these promising biofungicides into a practical, economically viable, and environment-friendly control strategy that can be incorporated into any integrated pest management program.

Background

Diseases caused by plant pathogens such as fungi and bacteria are one of the significant contributors to post-harvest losses (Gustafson 2019). Fungal phytopathogens are the most common and destructive causal organisms of plant diseases from pre- to post-harvest stages. Their persistence in different crop production systems is primarily attributed to their complex and varied characteristics (Palm 2001). Lasiodiplodia, Colletotrichum, and Fusarium are amongst the notorious genera that cause many post-harvest diseases of different crops (Coates and Johnson 1997).

To combat these pathogens, farmers have been dependent on the rapid action and efficiency of chemical fungicides (McGrath 2004). However, it has been well documented that most of these chemical residues are highly toxic and persist for years in the soil (Mahmood et al. 2016). Several studies were done to develop alternative approaches to manage fungal plant pathogens efficiently and sustainably for these reasons. Biological control of fungal plant pathogens, using bacterial antagonists, has been an alternative management strategy against these organisms. Several bacterial species under the genera Bacillus (El-Bendary et al. 2016), Pseudomonas (Weller 2007) and Serratia (Someya et al. 2000) are known to be inhibitory against some significant fungal plant pathogens.

Another group of antagonistic bacteria studied against plant pathogens is the entomopathogenic bacteria (EPB), which comprises species that are used for insect management programs. Recent studies have added Photorhabdus and Xenorhabdus spp., endosymbiotic bacteria of entomopathogenic nematodes (EPNs), Steinernema and Heterorhabditis, respectively, on the long list of antagonistic bacteria against many fungal plant pathogens (Fang et al. 2014) such as Glomerella cingulata, Phomopsis sp., Phytophthora cactorum, Fusicladosporium effusum, Monilinia fructicola (Shapiro-Ilan et al. 2009), some Colletorichum spp. (Bock et al. 2013), F. carpophylium and F. effusum (Hazir et al. 2016). These bacterial endosymbionts play a significant part in the capability of the EPNs in controlling insect pests (Tofangsazi et al. 2012). Upon entry of the EPNs to their respective hosts, these bacterial endosymbionts are released into the insect hemocoel to provide the EPNs enough nutrition for their growth and development. Part of the EPNs successful invasion is their secondary metabolites, containing either insecticidal toxins or anti-microbial proteins or even both. Some of these proteins are used to prevent the insect cadaver's immediate putrefaction and hamper other microbial organisms from infecting the cadaver (Muangpat et al. 2017).

This research is a pioneering Photorhabdus bioefficacy study in the Philippines, designed to determine the in vitro antifungal effect of local Photorhabdus strains against postharvest pathogens, L. theobromae, F. oxysporum f. sp. lycopersici, and C. species.

Methods

For the isolation of the EPB, 200 infective juveniles of the 2 nematode isolates, Heterorhabditis indica BSDS and H. indica MAP, were used to infect 13-day-old larvae of Ostrinia furnacalis (Guenee) (Lepidoptera: Carambidae). After approximately 24 to 48 h. post-infection, the larva's proleg was cut off to extract its hemolymph, which was then streaked onto nutrient agar supplemented with bromothymol blue and triphenyltetrazolium chloride (NBTA). The plates were incubated for 48 to 72 h. until pure green colonies appeared. These were purified further and grown in tryptic soy broth (TSB) and incubated at 28 °C for 24–72 h. in a shaking incubator. Culture stocks were prepared in 50% glycerol solution for long-term storage at − 80 °C.

Colony PCR was performed on Quanta Biotech S96 thermal cycler (Quanta Biotech, Ltd., Surrey, England) using the following programs: i) 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 12 min, 72 °C for 10 min, and 25 °C for 1 min and ii) 95 °C for 3 min, 25 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and 72 °C for 5 min, for Txp40 gene-based Photorhabdus (Brown et al. 2005) detection and 16s-based species identification (Jang et al. 2011), respectively. A 25-μl reaction mix for both markers composed of 12.5 μl of 2 × Taq Master Mix (Vivantis), 1.0 μl of forward and reverse primer (50 pmol), 0.25 μl of MgCl₂ (1.5 μM), 7.25 μl nuclease-free water (Vivantis), and 3.0 μl DNA template were used. The amplicons produced were then subjected to gel electrophoresis for 35 min at 100 V using a GelRed® (Biotium)-stained 1% agarose gel submerged in 0.5X TBE buffer. The amplicons were viewed under the AlphaImager Mini System (Protein simple, San Jose California, USA). 16s amplicons were sent to Apical Scientific Sdn, Bhd. (Malaysia) for Sanger sequencing. Sequences were edited using Bioedit software (Hall 1999) and subjected to megaBLAST for identity mining.

For collecting cell-free culture filtrates (CFC), tubes containing 10 ml TSB were inoculated with a loopful of each bacterial isolate and placed in an incubator shaker under 170 rpm at 28 °C for 48–72 h. The bacterial samples' optical density was measured using a spectrophotometer with a wavelength of 600 nm and adjusted to OD₆₀₀ = 2.0. The bacterial samples were then centrifuged at 15,000×g, at 4 °C for 15 min, and the supernatant was collected and passed through a 0.22 µm bacterial filter. The collected CFCs were mixed with PDA (25% v/v) and plated in 60 × 15 mm Petri plates. For the control, uninoculated TSB was added to PDA. With a sterile 0.7 cm cork borer, agar discs were cut off from the 5- to 7-day-old F. oxysporum f. sp. lycopersici, L. theobromae, and 2 Colletotrichum spp. cultures, and transferred onto the middle of the PDA plates. The fungal growth's actual diameter was measured for 2–7 days as soon as the control fungus reached the plate's edge. The data were subjected to analysis of variance (ANOVA), followed by Least Significant Difference post hoc test (LSD test), using Rstudio software (V. 4.0.2).

Results

Photorhabdus colonies were verified through the amplification of the Txp40 toxin gene marker of approximately 1200 bp. The amplified product agrees with the published amplicon sizes specific for this gene. Further characterization of the Photorhabdus BSDS and Photorhabdus MAP revealed their identity as P. luminescens based on 16s sequences with 99.51 and 99.58% similarity, respectively, to P. luminescens subsp. akhurstii (Accession No. AY278643.1) in the GenBank-NCBI database. The 16s sequence data of Photorhabdus BSDS and Photorhabdus MAP were uploaded to the GenBank-NCBI database with Accession No. MT658664 and MT658665, respectively.

Based on the bioassay experiment, independent analyses showed significant differences in percentage growth inhibition on PDA plates amended with CFCs of both P. luminescens BSDS and P. luminescens MAP compared to those grown without CFCs. The percentage growth inhibition of L. theobromae, Colletotrichum sp. and C. musae exposed to P. luminescens BSDS (60.51 ± 2.04, 74.15 ± 0.54, and 93.18 ± 0.46%, respectively) were significantly higher than those of P. luminescens MAP (37.50 ± 2.18, 30.11 ± 1.43, and 88.64 ± 0.66%, respectively). However, a different trend of growth inhibition caused by both CFCs was observed on F. o. f. sp. lycopersici with P. luminescens MAP (21.87 ± 0.71%) being more inhibitory than P. luminescens BSDS (9.95 ± 3.36). Relatively, the weakest inhibitory activity from both CFCs was observed against F.o. f.sp. lycopersici, while the most substantial effect can be seen on C. musae (Table 1, Fig. 1).

Table 1 Mean percentage growth inhibition of the tested plant pathogenic fungi on potato dextrose agar (PDA) plates treated with CFC of Photorhabdus luminescens BSDS and P. luminescens MAP

Fig. 1

Anti-fungal activity of Photorhadus luminescens BSDS and P. luminescens MAP cell-free culture filtrates against A Fusarium oxysporum f. sp. lycopersici at six days post-infection (dpi); B Colletotrichum sp. at five dpi; C Colletotrichum musae at four dpi; and D Lasiodiplodia theobromae at two dpi on PDA media. The control plates were amended with tryptic soy broth only

Discussion

In this study, CFCs of the bacterial isolates were used for the biological assay instead of the live cells because of the innate phase variation they possess during in vitro culture (Han and Ehlers 2001), which significantly affects anti-microbial metabolite production (Bock et al. 2013). In a similar study, CFCs from phase I variants of P. luminescens isolated from H. megidis and grown in TSB completely inhibited the growth of the fungal plant pathogens, Botrytis cinerea, Ceratocystis ulmi, Ceratocystis dryocoetidis, Mucor piriformis, Pythium coloratum, Pythium ultimum, and Trichoderma pseudokingii (Chen et al. 1994).

Furthermore, the results also indicated that there exists an intraspecific variation in terms of their antifungal effect. Variable mean growth diameter can be caused by the differences in toxicity, forms, and dosages of secondary metabolites found in the CFCs and the fungal phytopathogens' innate sensitivity. Shapiro-Ilan et al. (2009) also demonstrated the suppressive effects of Photorhabdus metabolites against pecan and peach fungal pathogens. In their study, 2 P. luminescens strains from 2 different H. bacteriophora strains, Hb and VS, also displayed variable effects with P. luminescens VS, causing a significantly higher growth inhibition to the pathogens, Glomerella cingulata, Monilinia fructicola, Phomopsis sp., Phytophthora cactorum, and Fusicladosporium effusum. Since the cell-free filtrate is a repertoire of many other metabolites, this heterogeneity might have also affected the specific anti-fungal metabolites' efficacy. Antimicrobial compounds in pure form are sometimes preferred over crude extracts. Some of the noteworthy metabolites of Photorhabdus that have been studied and proven to have antifungal activity in pure form in vitro include stilbene derivatives (Shi et al. 2017), trans-cinnamic acid (Bock et al. 2013), and benzaldehyde (Ullah et al. 2015).

Conclusions

The in vitro inhibitory activity of local P. luminescens strains against L. theobromae, F. oxysporum f. sp. lycopersici, and C. species is only a preliminary proof of their biological control potential. Further bioefficacy assays coupled with the proper formulation and more in-depth molecular and biochemical studies could lead to the development of sustainable Photorhabdus-based biotechnology with a great utility to any integrated disease management programs in the Philippines.