Control Potentials of Three Entomopathogenic Bacterial Isolates for the Carob Moth, Ectomyelois ceratoniae (Lepidoptera: Pyralidae) in Pomegranates

Abstract

Ectomyelois ceratoniae (Lepidoptera: Pyralidae) is the primary pest of pomegranates in Saudi Arabia and is mostly controlled using broad-spectrum pesticides. Providing environmentally sound choices to limit reliance on chemical management is a major challenge in the control of E. ceratoniae and, as a consequence, in the protection of pomegranate crops from its invasion. Entomopathogenic bacteria (EPB) symbiotically associated with entomopathogenic nematodes (EPNs) are well-known biocontrol agents of soil-dwelling or aerial pests. The bacterium symbiont (EPB) is the real insect-killing biocontrol agent, while the nematode (EPN) serves as a vector. We wondered whether the EPB vector, which is extremely vulnerable to adverse environmental conditions, like drought, high temperatures, and repellent soil microorganisms, could be omitted. We intended to evaluate the biocontrol potential of directly applied EPB cells and cell-free culture media (CFCM) on the larval instar E. ceratoniae. Xenorhabdus budapestensis DSM 16342 (EMA), X. szentirmaii DSM 16338 (EMC), and Photorhabdus luminescens ssp. laumondi (TT01) strains were used. After three days of exposure, the cells of EMA, EMC, and TT01 strains resulted in 100%, 88%, and 79.3% larval mortality rates, respectively. The applied EMA CFCM resulted in 53.7% larval mortality, indicating the presences of (at least) one extremely strong component produced by EMA. We concluded that the direct application of either the EPB cells or the CFCM must be a prospective alternative biocontrol of E. ceratoniae, especially to protect the important fruit (pomegranate, Punica granatum) cultivars. Especially, newly identified local EPB isolates could be applied as bio-pesticides for integrated management practices or organic pomegranate production.

1. Introduction

Pomegranate (Punica granatum) is one of the oldest known fruits, as it is found in the writings and artifacts of many cultures and religions. Pomegranate is one of the Kingdom of Saudi Arabia’s most in-demand fruits. There are several pomegranate varieties grown in Saudi Arabia, but the Taify pomegranate supply remains the most popular. Pomegranate cultivation and its potential yield and quality are affected by many insect pests [1,2]. The carob moth (Ectomyelois ceratoniae; Lepidoptera: Pyralidae) is a polyphagus destructive fruit pest worldwide that can cause major problems for pomegranates. Economic loss caused by carob moths in the pomegranate fields of Iran is 30%–80% [3], while the yearly damage rate caused by E. ceratoniae on pomegranate fruits in Tunisia varies by 29%–72% [4]. The larvae-infested fruits either drop or remain on the trees. As larval activity is hidden, commercial insecticides are not efficient against this pest [5]. Pesticides are considered one of the main causes of the observed loss of biodiversity in agro-ecosystems. When pomegranates are severely treated with pesticides, residues are mainly concentrated in the fruits [6]. Given that these fruits are freshly consumed, contamination with pesticides is undesirable.

To achieve this demand, non-chemical pest control strategies are required, which need to be efficient, environmentally sound, and guarantee similar production yields. Biological pest control, alone or as part of an integrated pest management strategy, provides an alternative to chemical pesticides. Natural products derived from living organisms show tremendous chemical diversity and provide great potential for finding new classes of compounds with novel modes of action. Bacteria are microorganisms characterized by their ability to produce secondary metabolites (SMs) or small-molecule natural products. Many of these SMs exhibit a variety of biological activities and are of interest to agrochemical, food, and pharmaceutical industries.

Entomopathogenic nematodes (EPNs) are extensively operated as biocontrol agents against insect pests. Their efficiency is highly dependent on the balance between parasitic activities and the immunological response of the host [7]. EPNs belonging to the Steinernematidae and Heterorhabditidae (Rhabditida) families have received the most attention because they possess many of the attributes of effective biological control agents against a wide spectrum of insect and nematode pests [8,9,10]. Steinernematids are symbiotically associated with entomopathogenic bacteria (EPB) from the genus Xenorhabdus, and heterorhabditid nematodes are symbiotically associated with EPB from the genus Photorhabdus. These symbiotic bacteria multiply and rapidly kill insects within a day or two. The bacteria then convert the insect into suitable food for the nematodes and generate some antibiotics [11] and anti-feedants that preserve the dead insect from putrefaction, while the nematodes feed and reproduce in it. Satisfactory results on the effect of EPNs in the laboratory and in field conditions have been reported for controlling cherry fruit flies [12,13] and pomegranate aphid, Aphis punicae [14]. Xenorhabdus szentirmaii is a unique source of highly efficient antimicrobial peptides against almost all known plant pathogens [15]. The geography of Taif as a high-altitude region in Saudi Arabia, which includes high mountains, agricultural plates, and valleys, is so remarkable that the EPB fauna is expected to be very rich. Therefore, this study aimed to examine the larvicidal activity of three EPB strains that had not been screened for the control of carob moth, E. ceratoniae, in the laboratory.

2. Materials and Methods

2.1. Bacterial Strains

In this study, three strains of symbiotic bacteria included in the bioassay were provided from the Fodor Laboratory at Pannonia University, in Keszthely, Hungary. The symbiotic bacteria, including two strains of Xenorhabdus, X. budapestensis DSM 16342 (EMA) and X. szentirmaii DSM 16338 (EMC), and Photorhabdus luminescens (TT01), which were isolated from the EPNs Steinernema bicornutum, Steinernema rarum, and Heterorhabditis bacteriophora, respectively, were used for determining oral toxicity against E. ceratoniae larvae. The bacteria were regularly cultivated on LBTA (Luria Bertani agar) indicator plates (10 g of peptone, 5 g of yeast extract, 5 g of sodium chloride, 15 g of agar, 25 mg of bromothymol blue, 40 mg of 2,3,5-triphenyltetrazolium chloride and 1 L of distilled water (pH 6.8)) in the dark at 25 °C (Figure 1A,B). In preparing the bacterial filtrate, as an inoculum for a 100-mL culture, a single dark blue/red Xenorhabdus or dark green Photorhabdus colony was placed separately into test tubes containing 5 mL of LB liquid media. Exactly 100-mL aliquots of culture were shaken overnight at room temperature in 500-mL Erlenmeyer flasks before being transferred to flasks containing 400 mL of the same media and shaken at 200 rpm for five days. To obtain the supernatant and bacterial pellet, the multiplied bacterial culture was centrifuged (13,000 rpm for 30 min) at 4 °C. A 0.22 m Millipore filter was used to filter the supernatant to get a cell-free filtrate, while the pellet was resuspended in sterile distilled water. The filtrate was stored at 4 °C for further diluting with sterile distilled water to provide concentrations of 600, 400, 200, 150, 75, and 25 μL/mL. Adjustment of the bacterial cell suspension at OD₆₀₀ to 1.0 was performed using a spectrophotometer (SpectroStar Omega, BMC Labtech, Aylesbury, UK). A 10-fold serial dilution spread plate was used, and the bacterial suspension concentration was 10⁸ (CFU/mL). Three dilutions of each bacterial cell suspension were adjusted to 10⁸, 10⁶, and 10⁴ CFU/mL.

2.2. Insects

Carob moth (E. ceratoniae) cultures were established from larvae obtained from commercial pomegranate orchards in Taif, Saudi Arabia, during the growth seasons of 2020. Insect larvae were reared on a wheat bran diet (300 g of wheat bran, 50 g of sugar, 120 mL of water, 130 mL of glycerol and 9 g of yeast), and adults were fed with a 10% honey–water solution. The rearing room was set at 25 ± 1 °C, 60% relative humidity and a 16 L:8 D light–dark cycle.

2.3. Bioassay for Larvicidal Activity

In the bioassays, late third and early fourth instar larvae were employed. Xenorhabdus and Photorhabdus suspensions were tested for toxicity against E. ceratoniae larvae. Each bioassay consisted of 25 larvae deposited in five wells of a 12-well plate (five larvae/well) provided with 5 g of pomegranate seeds as food for the larvae. Subsequently, 2 mL from each bacterial suspension or bacterial cell was distributed on the seeds in each well. Equal proportions of distilled water or sterile and filtered culture LB media were used as controls. The mortality rate of the larvae was observed after 12, 24, 48, and 72 h of exposure to bacterial suspensions or cells. On different dates, five replicates of each bioassay were examined. The experiment was repeated twice. When the larvae were teased with a fine sterile toothpick and no movement was detected, the larvae were pronounced dead. The larval mortality of E. ceratoniae after exposure to symbiotic bacteria was corrected following formula [16]:

$$\mathrm{Corrected}\mathrm{Mortality}\%=(\frac{\%\mathrm{MT}-\%\mathrm{MC}}{100-\%\mathrm{MC}})\times 100$$

where MT is mortality in treatment, and MC mortality in control.

The 50% lethal concentration (LC₅₀) and the 50% lethal time (LT₅₀) were determined by Probit analysis [17].

2.4. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

Chemical analysis of the bacterial filtrates was performed using GC-MS (GCMS QP2010 Ultra, Shimadzu, Kyoto, Japan). The GC column used was a Restek RT-2560 capillary column, which had 100% biscyanopropyl polysiloxane, length of 100 m, internal diameter of 0.25 mm ID, film thickness of 0.20 µm, fused silica and a highly polar phase, and was not bonded. The carrier gas was helium at a flow rate of 1 mL/min. The oven temperature was set to 130–145 °C, and the run time was 50 min. The purified samples were identified by comparing the mass spectra of the compounds with the library of the NIST 11 database (version 2.0, U.S. Department of Commerce, Gaithersburg, MD, USA).

2.5. Statistical Analysis

The corrected mortality percentage of the larvae was analysed using a two-way analysis of variance followed by Duncan’s multiple range test. All analyses were conducted using the Costat program. p-values less than 0.05 were considered significantly different. Data of the LC₅₀ and LT₅₀ were analysed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). The data obtained were expressed as mean ± standard error.

3. Results

3.1. Larvicidal Activity of Bacterial Filtrates on E. ceratoniae

Table 1. Larvicidal activity of three bacterial species filtrates on the carob moth, Ectomyelois ceratoniae under laboratory condition.

Bacterial Species	Concentration (µL mL−1)	a Corrected Mortality %				Bacterial Species Means
		12 h	24 h	48 h	72 h
EMA	600	b 80 ± 0	88 ± 4.9	95.7 ± 4.34	100 ± 0	53.7 a
	400	64 ± 4	72 ± 4.9	78.3 ± 0	86.98 ± 5.3
	200	48 ± 4.9	56 ± 4	60.86 ± 4.36	73.92 ± 8.14
	150	40 ± 0	47.82 ± 5.3	48 ± 4.9	56.5 ± 0
	75	26.1 ± 5.3	28 ± 4.9	30.4 ± 4.35	32 ± 4.9
	25	8 ± 4.9	21.7 ± 5.3	21.7 ± 5.3	24 ± 4
EMC	600	64 ± 4	72 ± 4.9	86.98 ± 5.3	91.32 ± 5.3	43.9 b
	400	48 ± 4.9	60 ± 0	65.2 ± 5.34	78.3 ± 6.88
	200	36 ± 4	48 ± 4.9	52.2 ± 4.34	69.6 ± 5.34
	150	32 ± 4.9	40 ± 0	47.82 ± 5.3	47.82 ± 5.3
	75	16 ± 4	21.7 ± 5.3	24 ± 4	26.1 ± 5.3
	25	0 ± 0	7.8 ± 3.2	8 ± 4.9	13.04 ± 0
TT01	600	36 ± 7.5	48 ± 4.9	65.2 ± 5.34	78.3 ± 0	26.7 c
	400	20 ± 0	32 ± 4.9	34.8 ± 0	56.5 ± 6.88
	200	20 ± 0	26.1 ± 5.3	28 ± 4.9	34.8 ± 0
	150	16 ± 4	17.4 ± 4.35	20 ± 6.3	34.8 ± 0
	75	8 ± 4.9	12 ± 4.9	13.04 ± 0	21.7 ± 5.3
	25	0 ± 0	4 ± 4	5.2 ± 3.2	7.8 ± 3.2
LB		0 ± 0	0 ± 0	0.87 ± 0.53	0.87 ± 0.53	0.43 d
Exposure duration Means		23.5 d	29.8 c	32.5 b	38.9 a

^a Each treatment in this experiment was represented by five replicates, each with five larvae insect. ^b Numbers in each column indicated to corrected mortality ± standard error. Means with different letters within the same column or row differ significantly (p < 0.05 using Duncan’s Multiple Range Test).

shows that the larvae of E. ceratoniae were highly susceptible (p < 0.05) to both species of Xenorhabdus among all concentrations and exposure periods tested, with averaged corrected mortality percentages of 53.7% and 43.9% for EMA and EMC, respectively. TT01 bacteria were less effective, as they induced a larval mortality rate of only 26.7% (p < 0.05). The mortality rate (p < 0.05) of the positive control (pomegranate seeds treated with the media alone) was 0.43%. The percentage mortality was likewise observed to have a direct association with the bacterial supernatant concentration (p < 0.05). Thus, as the concentration of supernatant increased, the mortality percentage increased. The mortality of E. ceratoniae larvae was significantly high (p < 0.05) with value of 57.2% when a bacterial cell-free suspension was used at a concentration of 600 µL mL⁻¹. Exposure time significantly affected the percentage of mortality (p < 0.05), as 38.9% larval mortality was recorded 72 h post-exposure. As shown in Table 1, EMA, EMC, and TT01 bacteria at concentrations of 25–600 µL mL⁻¹ caused larval mortality (Means ± SE) ranging from 8 ± 4.9% to 100 ± 0%, 0 ± 0% to 91.3 ± 5.3%, and 0 ± 0% to 78.3 ± 0%, respectively (p < 0.05). No significant difference was found between the two Xenorhabdus species in larval mortality after a 72-h exposure (p < 0.05), indicating a closer mean mortality of 100 ± 0% for EMA and 91.3 ± 5.3% for EMC. We reported high larval mortality rates for high concentrations of EMA and EMC bacteria for all counting days, as well as considerable mortalities due to TT01. Although EMA at a low concentration (25 µL mL⁻¹) showed remarkable larval mortality for all revised days, no significant difference was found in the mortalities due to EMC, TT01, and control (absence of natural products derived from bacteria) treatments, specifically at a 12-h exposure (0 ± 0%).

3.2. Larvicidal Activity of Bacterial Cells on E. ceratoniae

As shown in

Table 2. Larvicidal activity of three bacterial species cells on the carob moth, Ectomyelois ceratoniae under laboratory condition.

Bacterial Species	Concentration (CFU mL−1)	a Corrected Mortality %				Bacterial Species Means
		12 h	24 h	48 h	72 h
EMA	108	b 84 ± 4	88 ± 4.9	100 ± 0	100 ± 0	81.6 a
	106	72 ± 4.9	80 ± 0	88 ± 4.9	92 ± 4.9
	104	56 ± 4	60 ± 0	71.3 ± 4.6	87.7 ± 5.05
EMC	108	72 ± 4.9	80 ± 0	83.7 ± 4.1	88 ± 4.9	67.4 b
	106	52 ± 4.9	60 ± 8.9	66.96 ± 5.02	79.3 ± 0.4
	104	48 ± 4.9	52 ± 4.9	54.96 ± 7.1	71.3 ± 4.6
TT01	108	40 ± 6.3	40 ± 0	67.3 ± 4.5	79.3 ± 0.4	42.8 c
	106	28 ± 4.9	44 ± 7.5	50.96 ± 7.5	58.96 ± 6.1
	104	8 ± 4.9	20 ± 8.9	34.3 ± 6.8	42.3 ± 3.7
Exposure duration Means		51.1 d	58.2 c	68.6 b	77.6 a

^a Each treatment in this experiment was represented by five replicates, each with five larvae insect. ^b Numbers in each column indicated to corrected mortality ± standard error. Means with different letters within the same column or row differ significantly (p < 0.05 using Duncan’s Multiple Range Test).

, the same trend was recorded regarding the effect of bacterial cells on the mortality of E. ceratoniae larvae. The data also indicated that larval mortality was strongly dependent on the bacterium species, concentration of bacterium cells, and exposure duration (p < 0.05). According to the results, EMA surpassed EMC and TT01 in inducing mortality in E. ceratoniae among all concentrations and exposure periods tested. EMA induced larval mortality of 81.6% compared with the 67.4% and 42.8% induced by EMC and TT01, respectively. EMA bacterial cells at a higher concentration (10⁸) were more effective than EMC, as they had the ability to induce 84 ± 4%, 88 ± 4.9%, 100 ± 0%, and 100 ± 0% mortalities of the late third instar larvae of E. ceratoniae at 12, 24, 48, and 72 h of application, respectively, compared with the 72 ± 4.9%, 80 ± 0%, 83.7 ± 4.1%, and 88 ± 4.9% mortalities induced by EMC at 12, 24, 48, and 72 h of application, respectively. The results showed that all bacterial species cells at a low concentration (10⁴), except for TT01, induced considerable larval mortality rates for all exposition periods ranging from 56 ± 4% to 87.7 ± 5.1%, 48 ± 4.9% to 71.3 ± 4.6%, and 8 ± 4.9% to 42.3 ± 3.7% for EMA, EMC, and TT01, respectively (Table 2).

3.3. Comparison of the Effect of the Bacterial Filtrate and Bacterial Cells on the Larval Mortality of E. ceratoniae

Figure 2 shows that the cell-free supernatant and the cells of all tested bacteria had a highly significant effect on the mortality of E. ceratoniae larvae at 72 h of exposure (p < 0.0001). The application of bacterial cells induced a more significant (F = 176.5; p = 0.0001; df = 24) larval mortality of E. ceratoniae, with a mean mortality rate of 77.6%. Conversely, the bacterial cell-free supernatant had a lower virulence, with a mortality rate of 51.7%. Moreover, the results indicated that the mean percentage of larval mortality caused by EMA (77.4%) was significantly (F = 69.03; p = 0.0001; df = 24) higher than that caused by EMC (66.9%) and TT01 (49.6%). The interactive effect of the bacterium species and the application method on larval mortality was not significant (F = 2.439; p = 0.1086; df = 24).

3.4. Lethal Concentration and Time of the Bacterial Filtrate and Cells in the Larval Mortality of E. ceratoniae

As shown in

Table 3. Lethal concentration and lethal time of bacterial species against third instar larvae of E. ceratoniae.

Bacterial Species	a LC50		LT50 (h)
	Filtrate (µL mL−1)	Cells (CFU mL−1)	Filtrate	Cells
EMA	125.9	2.14 × 102	19.95	5.25
EMC	204.2	1.78 × 103	52.48	7.36
TT01	575.4	8.13 × 106	208.93	44.57

^a LC₅₀ and LT₅₀: lethal concentration and exposure time kills 50% of insect host. Concentration expressed as µL/mL distilled water for bacterial cell-free supernatant and CFU/mL distilled water for bacterial cells.

, the calculated LC₅₀ values in the carob moth larvae under laboratory conditions were 125.9 µL/mL cell-free supernatant and 2.14 × 10² CFU/mL, 204.2 µL/mL and 1.78 × 10³ CFU/mL, and 575.4 µL/mL and 8.13 × 10⁶ CFU/mL for the EMA, EMC, and TT01 bacteria, respectively. Moreover, the lethal time of larval mortality was lower in the bacterial cell application than in the bacterial filtrate application. The EMA and EMC cells were more virulent against E. ceratoniae larvae, as they recorded lower LT₅₀ values of 5.25 h and 7.36 h, respectively. The TT01 cells had an LT₅₀ of 44.57 h. The lethal times of the bacterial filtrates were 19.95, 52.48, and 208.93 h for EMA, EMC, and TT01, respectively.

3.5. GC-MS Analysis of the Bacterial Cell-Free Supernatant

Table 4. The chemical composition of Xenorhabdus szentirmaii DSM 16338 (EMC).

No.	R. Time (min)	Name	Formula
1	9.265	3.beta.,17.beta.-dihydroxyestr-4-ene	C18H28O2
2	9.670	1-Methylcyclopropanemethanol Heptanal, 2-methylPiperazine, 2-methyl	C5H10O C8H16O C5H12N2
3	9.973	1-Propanol, 3-ethoxy	C5H12O2
4	10.295	Oxirane, tetramethyl-	C6H12O
5	10.424	Cyclohexanol, 4-methyl-, cis	C7H14O
6	11.478	1-Methoxy-3-hydroxymethylheptane	C9H20O2
7	12.900	Nonanal	C9H18O
8	13.524	4,4-Dimethoxy-2-methyl-2-butanol	C7H16O3
9	17.035	N-(4-Methylcyclohexyl)acetamide	C9H17NO
10	17.277	4-Cyclohexene-1,2-diol	C6H10O2
11	18.257	Thiirane	C2H4S
12	19.717	Oleic Acid $$ 9-Octadecenoic acid (Z)	C18H34O2
13	20.052	2-Decenal, (Z)-	C10H18O
14	22.714	E-2-Tetradecen-1-ol	C14H28O
15	23.156	1,1-Dodecanediol, diacetate	C16H30O4
16	25.945	Hexadecanal	C16H32O
17	29.306	Octadecanal $$ Stearaldehyde	C18H36O
18	41.858	1-Formyl-3-methylaziridine-2-carbonitrile	C5H6N2O

,

Table 5. The chemical composition Xenorhabdus budapestensis DSM 16342 (EMA).

No.	R. Time (min)	Name	Formula
1	9.25	3.beta.,17.beta.-dihydroxyestr-4-ene	C18H28O2
2	9.669	1-Methylcyclopropanemethanol Heptanal, 2-methylPiperazine, 2-methyl	C5H10O C8H16O C5H12N2
3	9.97	4,4-Dimethoxy-2-methyl-2-butanol 2-Butanol, 1-methoxy	C7H16O3 C5H12O2
4	10.294	Oxirane, 3-ethyl-2,2-dimethyl Oxirane, tetramethyl	C6H12O C6H12O
5	10.424	Heptanal $$ n-Heptaldehyde	C7H14O
6	11.476	Octanal $$ n-Caprylaldehyde	C8H16O
7	12.896	Nonanal	C9H18O
8	13.523	4,4-Dimethoxy-2-methyl-2-butanol Cycloserine	C7H16O3 C3H6N2O2
9	17.022	N-(4-Methylcyclohexyl)acetamide	C9H17NO
10	17.277	4-Cyclohexene-1,2-dio	C6H10O2
11	19.706	Oleic Acid	C18H34O2
12	20.047	2-Decenal, (Z)-	C10H18O
13	22.703	Pentadecanoic acid	C15H33O2
14	23.151	2-Undecenal	C11H20O
15	25.942	10-Methyl-E-11-tridecen-1-ol propionate	C17H32O2
16	26.461	Z-8-Methyl-9-tetradecenoic acid	C15H28O2
17	26.753	Cyclopentaneundecanoic acid	C16H30O2
18	29.3	Tetradecanal	C14H28O2
19	29.781	3-Acetoxydodecane	C14H28O2
20	38.122	12-Methyl-E,E-2,13-octadecadien-1-ol 8-Hexadecenal, 14-methyl-, (Z)-	C19H36O C17H32O
21	41.872	1-Formyl-3-methylaziridine-2-carbonitrile	C5H6N2O

and

Table 6. The chemical composition of Photorhabdus luminescens (TT01).

No.	R. Time min	Name	Formula
1	9.29	3.beta.,17.beta.-dihydroxyestr-4-ene	C18H28O2
2	9.698	1-Methylcyclopropanemethanol Heptanal, 2-methylPiperazine, 2-methyl	C5H10O C8H16O C5H12N2
3	10.00	4,4-Dimethoxy-2-methyl-2-butanol 2-Butanol, 1-methoxy	C7H16O3 C5H12O2
4	10.325	Oxirane, 3-ethyl-2,2-dimethyl Oxirane, tetramethyl	C6H12O C6H12O
5	10.452	Heptanal	C7H14O
6	11.507	Octanal $$ n-Caprylaldehyde	C8H16O
7	12.923	Nonanal	C9H18O
8	13.55	2-Amino-1,3-propanediol	C3H9NO2
9	16.206	Propanamide, 2-hydroxy-N-methyl-	C4H9NO2
10	17.045	Cyclopentaneundecanoic acid N-Cyclododecylacetamide	C16H30O2 C14H27O2
11	17.229	2-Nonenal, (E)-9-Octadecenoic acid (Z)-, hexyl ester	C9H16O C24H46O2
12	18.066	6-(Hydroxy-phenyl-methyl)-2,2-dimethylcyclohexanone	C15H20O2
13	20.066	2-Decenal, (Z)	C10H18O
14	23.164	2-Undecenal	C11H20O
15	25.957	E-2-Tetradecen-1-o	C14H28O2
16	26.487	Z-10-Tetradecen-1-ol acetate	C16H30O2
17	26.763	Cyclopentaneundecanoic acid 1-Hexadecyne	C16H30O2 C16H30O
18	29.313	Octadecanal $$ Stearaldehyde	C18H36O
19	29.760	Octanoic acid, 7-oxo-	C8H14O3
20	36.091	9-Hexadecenoic acid	C16H30O2
21	38.166	12-Methyl-E,E-2,13-octadecadien-1-ol	C19H36O
22	38.469	7-Methyl-Z-tetradecen-1-ol acetate	C17H32O2
23	41.836	1-Formyl-3-methylaziridine-2-carbonitrile	C5H6N2O

show the chemical compositions of three different samples of bacterial filtrates. The samples were run using GC-MS, which identified different substances in one test sample, and the components of the mixtures were separated and analysed. GC-MS has the capacity to resolve sample extracts containing hundreds of compounds. During the analysis process, samples are ionized and fragmented by the mass spectrometer, and the ions are separated based on their different mass-to-charge (m/z) ratios. GC-MS data acquisition can be performed to cover a wide range of m/z ratios. Using extensive commercially available libraries of mass spectra, unknown compounds and target analytes can then be identified and quantified accurately. Table 4, Table 5 and Table 6 show the retention time, chemical name, and chemical formula of three bacterial filtrate samples. Similarities were found among the three samples. For example, compounds 1, 2, and 3 were the same in the three bacterial species. Moreover, the compounds retained at 12.89–12.92 min and the samples retained at 20.04–20.06 min and at 41.83–41.85 min were the same in the three bacterial filtrates. Some similarities appeared in only two species: compounds 9 and 10 in EMC were the same as compounds 9 and 10 in EMA, compound 14 in EMC was the same as compound 15 in EMA, and compound 20 in EMA was the same as compound 21 in TT01. Some compounds were identified in only one species, such as compound 11 in EMC. Thus, by comparing the retention time and the chemical name, we found that the three bacterial species were similar, especially the EMA and EMC samples, as shown in the results in Table 4 and Table 5.

4. Discussion

In this study, we demonstrated the toxicity of the cell-free supernatant or the cells of three bacterial species in the late third and early fourth instar larvae of the carob moth E. ceratoniae under laboratory conditions. The results revealed that all the bacterial species tested successfully induced mortality in E. ceratoniae larvae. The mortality % was directly related to the exposure period and bacterial concentration if they were applied as the cell-free supernatant or cells. Moreover, the larvae of E. ceratoniae were more susceptible to bacterial cell infestation than the bacterial cell-free supernatant. These results were consistent with those of Yooyangket et al. [18], who supposed that symbiotic bacteria become virulent when ingested by mosquito larvae. The findings also revealed that the two species of Xenorhabdus were more effective than the Photorhabdus species against E. ceratoniae larvae. This higher lethality of Xenorhabdus than Photorhabdus correlated with the better efficacy of Steinernema carpocapsae and Steinernema feltiae compared with H. bacteriophora, which showed low virulence on carob moth larvae in laboratory conditions [19]. Conversely, Elbrense et al. [20] found that Photorhabdus sp. was more effective against Pieris rapae and Pentodon algerinus larvae than Xenorhabdus sp. Salvadori et al. [21] reported that the Photorhabdus species killed 75–96% of Spodoptera frugiperda larvae, while Xenorhabdus bacteria were less active, causing 33%–57% mortality.

Our results revealed that the maximum larval mortality was 100% after 72 h of exposure to 600 µL/mL EMA cell-free supernatant and 10⁸ CFU/mL after 48 and 72 h. Similarly, after three days of exposure, we found a larval mortality rate of over 65% for EMC cell application, but the larval mortality rate did not reach 50% when the cell-free supernatant was applied. These results were consistent with those of Fukruksa et al. [22], who reported that the cell suspensions of Xenorhabdus ehlersii isolated from Steinernema scarabaei were found to be efficient at killing 100% of Ae. aegypti. Conversely, E. ceratoniae larvae had a low mortality rate (8 ± 4.9%) after exposure to P. luminescens (TT01) for 12 h at a concentration of 75 µL/mL cell-free supernatant or 10⁴ CFU/mL. This could be attributed to its failure to produce toxic metabolites to E. ceratoniae. Various symbiotic bacteria strains can produce a variety of secondary metabolites, resulting in varying levels of efficacy in eliminating target organisms. Furthermore, the test of insect mortality rate may be related to the variable number of bacterial cells ingested by each larva. The fact that both Xenorhabdus sp. and Photorhabdus sp. bacteria produce toxin complexes, proteases, lipases, lipopolysaccharides, and other active components may explain their insecticidal activity [23]. These compounds cause caterpillars to become floppy [24], induce apoptosis, inhibit haemocyte motility, and suppress cellular and humoral immunity [25]. Xenorhabdus and Photorhabdus species devote a large portion of their resources to the production of specialized metabolites generated from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria form a symbiotic relationship with nematodes, followed by an insect pathogenic phase [26]. At the DNA sequence level, both bacteria appear to be almost identical, indicating a close evolutionary relationship. However, high-resolution mass spectrometry investigations show that the two taxa contain a wide range of chemical compounds. The xefoampeptides [26,27] and tilivalline [28] were among the previously unknown metabolite groups discovered through molecular network reconstruction.

At least eight bacterial metabolites are produced by Xenorhabdus nematophila that play central functions in destroying and inhibiting the immune reactions of the targeted insects [29]. Toxin complexes (Tcs), make caterpillars floppy (Mcf), Photorhabdus insect-related proteins, and Photorhabdus virulence cassettes have been reported to be produced by Photorhabdus species [30]. The Tcs damage the insect’s midgut epithelial cells, but the Mcf is active immediately after injection and disturbs the insect’s haemocytes by causing apoptosis [31]. The GC-MS analysis of our EPBs revealed that the Xenorhabdus bacteria possessed 21 main components, while the Photorhabdus bacteria had 23 main components. Six of these compounds (3.beta,17.beta.-dihydroxyestr-4-ene, 1-methylcyclopropanemethanol heptanal, 2-methylpiperazine, 2-methyl, oxirane tetramethyl, nonanal, 2-decenal (Z), and 1-formyl-3-methylaziridine-2- carbonitrile) were commonly found in the three bacterial species. Moreover, the levels in the two species of Xenorhabdus were higher than those in the Photorhabdus species. Similarly, Hasan et al. [32] attributed the virulence of six X. nematophila strains against Spodoptera exigua to active secondary substances, such as benzeneacetic acid, n-decanoic acid, tetradecane, 1-decene, and 3-benzylidene-hexahydro-pyrrolo, which suppress the immune system of insects. Mollah and Kim [33] also detected fatty alcohol, 1-ecosine, heptadecane, octadecanes, and methyl-12-tetradecen-1-ol acetate in different strains of Xenorhabdus and Photorhabdus bacteria. The authors suggested that these compounds inhibited insects’ phospholipase A2, thereby eradicating their immune system.

5. Conclusions

As E. ceratoniae is the most destructive pest in Saudi Arabia, there is an urgent need to provide tools to reduce its propagation, particularly in organic production, where the use of pesticides is strictly limited. To the best of our knowledge, no previous studies have related the direct application of EMA, EMC, and TT01 bacteria to control the same pest. On the other hand, for the first time, we verified the insecticidal activity of two Xenorhabdus and Photorhabdus bacterial cells or cell-free supernatants against E. ceratoniae larvae when ingested orally. Nevertheless, additional studies are required to evaluate the effect of EPB application on E. ceratoniae larvae in the field for a sustainable agro-environment.