New insights on entomopathogenic bacteria isolated from soil of citrus crops to combat the polyphagus aphid pest Hyalopterus pruni (Geoffroy 1762) (Hemiptera, Aphididae)

Abstract

Background

The employment of entomopathogenic microorganisms is a promising approach for ensuring optimal phytosanitary protection in the framework of biological management of insect crop pests. Among these microbes, entomopathogenic soil-borne bacteria are preferred over pesticides because they help successfully in the natural regulation of arthropod populations, as soil has a favorable ecology for the availability and richness of many beneficial bacterial species. In this study, it was focused on the isolation, identification and characterization of entomopathogenic bacteria isolated from cultivated citrus soils and on the evaluation of their insecticidal potential in the laboratory against the mealy plum aphid, Hyalopterus pruni (Geoffroy 1762) (Hemiptera: Aphididae), a polyphagous and major pest attacking and virus-transmitting of several Prunus crops.

Results

Isolation results gave 11 bacterial isolates, which were more than 50% effective in selection tests on H. pruni aphids at 1 × 10⁸ CFU/ml. A total of seven isolates generated significant insecticidal potential at different concentrations, and their molecular identification based on 16S rRNA genome sequencing yielded the following results: Lysinibacillus fusiformis (B4), Bacillus thuringiensis (B13), B. thuringiensis (B22), B. thuringiensis (B23), B. thuringiensis (B24), Pseudomonas sp. (P2) and Enterococcus gallinarum (P4). The most pathogenic potential of these strains was for E. gallinarum (P4), which induced 100% H. pruni mortality after 72 h of treatment at the concentration (C3 = 1 × 10⁶ CFU/ml), followed by B. thuringiensis (B23), (B22), (B4), (P2), (B13) and (B24) isolates, which caused 96, 91, 85, 83, 65 and 50% mortality rates, respectively, at the lowest concentration (C4 = 1 × 10⁵ CFU/ml). The LC₅₀ and LT₅₀ values were calculated for the entomopathogenic isolates of (P2), (P4), (B23) and (B24). A lowest LC₅₀ value was 1.08 × 10² CFU/ml for (P4) E. gallinarum after 72 h of treatment, whereas (P2) Pseudomonas sp. presented the shortest LT₅₀ of 33.6 h at concentration (C4 = 1 × 10⁵ CFU/ml).

Conclusions

The present study's outcomes have confirmed the existence, abundance, and variety of entomopathogenic bacteria at the soil level in citrus groves. Interestingly, these bacteria could be useful for aphids’ population control on a wide scale through the utilization of their toxins and enzymes, even against insect pests of a broad order.

Background

Insect pests are part of bio-aggressors that pose a significant risk to agricultural output, impacting the production level, quality, and aesthetic value of the crop. Aphids belong to the "sap-sucking" pests which about 100 species are considered severe agricultural and horticultural insect pests, directly responsible for destroying many economically significant crop plants. Population growth rate, considering the aphids’ age-specific development, survival, and fecundity, accurately characterizes this pests' impact (Blackman and Eastop 2000). Aphids can cause not only direct damage by sucking sap, but also indirect damage as vectors for various viral diseases. Prunus crops, such as peaches, are considered to be the best-known cultures in terms of their economic impact and production (Rousselin et al. 2017) worldwide. The mealy plum aphid Hyalopterus pruni (Geoffroy 1762) (Hemiptera: Aphididae), is a major polyphagous pest to several crops, including the genus Prunus globally growing under temperate and Mediterranean climates and in Europe and North America (Rousselin et al. 2017). H. pruni, causes damage directly to Prunus trees by absorbing phloem sap, reducing fruit yield quality and causing chlorosis which consequently affects growth in the long term (Lozier et al. 2009). This aphid species is also the main vector for plum pox virus (PPV) transmission which is responsible for shark disease, causing major economic losses on Prunus genus trees (Hazir et al. 2021).

Chemical insecticides are still widely used in peach orchards to control the aphid population. Despite those chemical substances targeted effect, insect populations may swiftly acquire resistance, unfortunately making these insecticides useless and impeding long-term management (Paliwal et al. 2022). Furthermore, detrimental effects on human health due to the indiscriminate pesticide usage, inhalation poisoning, toxicity in the food chain, contamination of surface and groundwater, and other environmental issues pose a serious obstacle to the ability to safeguard the quantity and quality of numerous important crops.

Exploring soil-borne bacteria diversity remains an attractive alternative to develop biological control of insect pests using these natural microorganisms (Meddas et al. 2020). Indeed, the soil is a site of microbial competition because of its vast biological diversity, including rhizospheric bacteria that influence plant development and most importantly control insect pests in a variety of crops. Bacteria of the Bacillus and Pseudomonas genera are considered to be the most widely used rhizospheric bacteria for potential biological control since they are capable of efficiently combating a wide variety of commercially important insect pests (Oulebsir-Mohand kaci et al. 2015a, b).

Bacteria of the Bacillus genus such as Bacillus thuringiensis (Bt) constitute approximately 95% of the global biopesticide economy, due to their extensive utilization as microbial control agents. The bacterium B. thuringiensis produces insecticidal proteins in the form of crystalline inclusions during the sporulation phase, known as Cry or Cyt toxins, which have proved effective against important lepidopteran pests. Some Bt strains produce a binary toxin called Vip2Ae-Vip1Ae, in addition to new Cry proteins from the Bt strain H1.5 named Cry41Ab1 and Cry41Aa1, respectively (Palma et al. 2014). Likewise, Pseudomonas is recognized for its entomopathogenic activity against a variety of insect pests, such as aphids (Manjula et al. 2018). P. fluorescens can lessen the severity of numerous fungal illnesses and improve Plant Growth Promotion Rhizobacteria (PGPR) by the synthesis of several secondary metabolites, such as hydrogen cyanides, siderophores, and antibiotics (O’Sullivan and O’Gara 1992).

Sustainable methods that use beneficial microorganisms’ natural insect diseases to reduce environmental harm, still become more meaningful to date. It has been determined that more than 100 bacterial species exhibiting entomopathogenic activity are exo- and endo-pathogens of arthropods. Aphid populations have been effectively controlled by entomopathogenic fungi, while bacterial control methods have achieved not much focus. This present investigation is based on the isolation and identification of rhizospheric bacteria from different soils cultivated on citrus, in order to investigate the insecticidal potential of effective isolates on the most damaging insect pest of Prunus, Hyalopterus pruni.

Methods

Soil sampling

Sampling techniques consisted of collecting sufficient soil volumes from four randomly selected locations in citrus groves. Soil samples were collected from the localities of Beni Mered, Mouzaïa, Oued El Alleug, Bougara, and Tabainat surrounding citrus area in the province of Blida (Central Mitidja plain, Algeria). Soils specimens were gathered from a depth of 10 to 15 cm near the root system, to isolate bacteria of the Pseudomonas genus (Bikram et al. 2018), and from a distance of 5 to 10 cm in the soil's uppermost stratum, in order to separate Bacillus strains (Mihir et al. 2017). Every soil sample collected was placed in hermetically sealed sterile plastic bags, until usage in the laboratory.

Bacteria isolation

Soil samples were dried separately, sieved, and finely ground. After that, 10 g of soil was suspended in 90 ml of sterile physiological water and then homogenized with a magnetic stirrer for 30 min. Successive dilutions were then prepared to obtain the final dilution of 10^–5 from a 10^–1 one. The dilutions used to isolate Bacillus bacteria were heated at 80 °C for 10 min to eradicate non-spore-forming bacteria, each prepared dilution was applied in 0.1 ml nutrient agar (NA). Pseudomonas, was incubated in a King B medium, which allows the production of fluorescein (Oulebsir-Mohand kaci et al. 2015a, b). Incubation was carried out at 30 °C for 24 h for Bacillus and at 28 ± 2 °C for 24 to 72 h in the case of Pseudomonas.

Identification of bacterial isolates

Morphological and biochemical characterization

The identification and taxonomic characterizations of isolated bacteria were based on several morphological characteristics corresponding mainly to the type of bacterial colony (size, shape, color, margin, opacity, and pigmentation), the shape of the bacterial cells, Gram staining and their mobility. Afterward, according to Bergey's Manual of Systematic Bacteriology using morphological, physiological, and biochemical methods (Buchanan and Gibbons 1974), the colonies that seem to meet the criteria of the desired genera (Bacillus and Pseudomonas) were successively transplanted in adequate medium to obtain a pure culture.

Molecular identification

Molecular identification focused on the sequencing of 16S rRNA genes for the performing strains being most efficient against the mealy plum aphid H. pruni at different selected concentrations. Bacterial genomic DNA was extracted using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies Sdn Bhd, Selangor DE, Malaysia). Isolated DNA was kept at 4 °C until needed for PCR. The amplification of the bacterial genomic extract was achieved using the primer set for the 16S rRNA gene (27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 1492R 5′-CCG TCA ATT CCT TTG AGT TT-3′) (Edwards et al. 1989) using a thermocycler (iCycler BioRad, USA). The runs were as follows: initial denaturation at 94 °C for 12 min, followed by other denaturation for 30 s at 94 °C, 30-s annealing at 55 °C, and 1 min 40 s for a primer extension at 72 °C. The amplification was repeated in 30 cycles, followed by a final extension at 72 °C (7 min).

PCR products were separated into a 1.5% agarose gel (Sigma-Aldrich, USA), and the gel was viewed under UV light after staining with Midori Green Advance (Nippon Genetics, Japan) and inspected using a UV trans-illuminator. PCR electrophoresis products were purified (Clean-Upkit, Vivantis) for sequencing onto a 3130 Genetic Analyzer Capillary Array for detection (Applied Bio systems) in the forward and reverse directions in separate reactions and duplicates. A BLAST analysis was conducted on the 16S rRNA sequence data by comparing similarity percentages using the NCBI GenBank database (NCBI GenBank; http://blast.ncbi.nlm.nih.gov) (Altschul et al. 1990). A phylogenetic tree was created using Molecular Evolutionary Genetics Analysis (MEGA 11) (Tamura et al. 2021).

Pathogenicity bioassays on H. pruni

Insect collection

The mealy plum aphid, H. pruni was collected from a non-treated peach orchard located in the experimental station at Saad Dahlab University Blida-1 (USDB-1), at the end of April 2022.

Preparation of bacterial suspensions

The isolated and characterized bacteria strains were purified on nutrient agar plates to get pure colonies for each isolate. After 24 h of incubation at 30 °C, a few colonies were collected and inoculated into 5 ml of nutrient broth to prepare the bacterial suspensions. The tubes containing bacterial solutions were incubated at 30 °C with 200 rpm shaking for 48 h to allow sporulation and crystal formation (Karungu et al. 2018). A volume of 1 ml of this bacterial solution was then seeded in tubes containing 5 ml of sterile distilled water. Bacterial concentrations used for bioassays were established by adjusting the optical density of the bacteria dilution at 625 nm to 0.1 (about 1 × 10⁸ CFU/ml) (Agbe et al. 2020).

Screening tests and bioassays

Adults H. pruni were firstly treated in a preliminary test in triplicate with a bacterial suspension concentration of 1 × 10⁸ CFU/ml, for all bacterial isolates (twenty-nine) in order to determine the most efficient isolate on aphids. Fresh peach leaves not infested with aphids were sterilized for two minutes with 2° chlorine bleach, then rinsed three times with sterile distilled water, dried with sterile absorbent paper and soaked on both sides for 2 min in the previously prepared bacterial suspension. Pathogenicity tests are based on soaking leaves in bacterial solutions, the way aphids feed by using their perforating mouthparts to penetrate plant tissues and extract sap from the phloem, ensuring that bacterial cells pass directly into the intestine. A number of 15 aphid individuals were then placed on the soaked leaves and put into sterile Petri dishes with wet piece cotton with sterile distilled water to maintain leaf vitality. The bacterial suspension was replaced with sterile distilled water for negative controls.

In further bioassay, isolates (11 isolates) that expressed more than 50% aphids mortality in the preliminary screening test were used at four different concentrations (1 × 10⁸, 1 × 10⁷, 1 × 10⁶ and 1 × 10⁵ CFU/ml). Treatments were performed with 15 aphid individuals per replicate and three replicates per concentration. Positive controls leaves were soaked in the same concentrations solutions of referenced strains Bacillus sp. HF911367 (Bt) and Pseudomonas sp. HF911366 (Pf) provided by laboratory of Valorization and Conservation of Biological Resources (VALCORE), M’Hamed Bougara University – Boumerdes (Algeria) (Oulebsir-Mohandkaci et al. 2015a, b) and negative controls infested leaves, were treated with sterile distilled water.

Mortality rate

Mortality rate was recorded after 24, 48, and 72 h, for all replicates both in screening tests and bioassays at different concentrations, based on the ratio of the total dead aphids number over the total aphids’ number used in treated and controls. The observed mortality was corrected using the ABBOT formula, (Abbott 1925): Percentage of corrected mortality MC% = 100 * (M1–M2/100-M1), where M1 was the percentage of mortality observed in controls and M2 was the percentage of mortality observed among treated.

Statistical analysis

To determine differences among treatment means, analysis of variance was performed using XLSTAT 2023V 1.6.1410 for Windows and The IBM SPSS Statistics 20 software. In the event of significant differences, Tukey's HSD test and Student's t test were used to separate the means of the different treatments. All parameters were analyzed at the 5% significance level. For controls Bt and Pf strains and bacteria isolates potentially efficient at different concentrations on H. pruni individuals, probit analysis was used to calculate median lethal concentrations and times (LC_50/90, LT_50/90) using Fisher and Yates method (1971) (Finney 1971).

Results

Morphological and biochemical characterizations

Isolation from the soil of citrus orchards in the Blida region resulted in a total of 90 isolates, including initially the two desired genera Bacillus and Pseudomonas. After 24 and 48 h of incubation for the respective isolates, some colonies were observed that developed on nutrient agar (NA) and King B. Colonies that probably belonged to the desired genus were selected. All isolates belonging to the Bacillus genus were large 2–4 mm colonies, cream-beige color, dry with irregular contour while being opaque (Fig. 1a). Pseudomonas, colonies were characterized by a regular contour, opaque, rather large, and round (Fig. 1b), with the typical production of fluorescent pigment.

Fig. 1

Colony morphological of Bacillus in nutrient agar medium (a) and Pseudomonas in King B (b)

Molecular identification

GenBank accession number of the 16S rRNA gene sequences for strains: B4, B13, B22, B23, B24, P2 and P4 is OR915493, OR915490, OR915491, OR915495, OR915489, OR915494 and OR915492, respectively. BLAST analysis of the five bacterial strains coded, B1, B2, B3, B4, and B5, indicated that they belong to the genus Bacillus (Bacillaceae family) with very similar sequence (100%) to Bacillus thuringiensis and Bacillus cereus (Table 1) (Fig. 2). The other isolates showed a 100% similarity sequence with other bacteria species. Strain B23 showed 100% similarity in his sequence with Bacillus proteolyticus, B24 and B13 with B. wiedmannii and B22 with strain B. anthracis. However, only two type strains, Lysinibacillus fusiformis and L. sphaericus, were closely linked to strain B6, which showed substantial sequence identity (100%). In other cases, P2 has a total similarity (100%) with Pseudomonas sp, whereas P4 belongs to the Enterococcus genus, and presented a high sequence similarity (100%) with the strain Enterococcus gallinarum strain GI13 (MT158590.1) and Enterococcus sp. (Table 1) (Fig. 2).

Table 1 Suggested identification of bacterial isolates, based on BLAST analysis of their 16S rRNA gene sequences

Fig. 2

Phylogenetic tree of selected isolates using neighbor-joining method based on 16S rRNA gene sequencing

In conclusion, bacterial isolates were identified as follows: B. thuringiensis for B23, B24, B13 and B22 isolate, Enterococcus gallinarum (P4), Lysinibacillus fusiformis (B4) and Pseudomonas sp. (P2) (Table 2). The phylogenetic position of bacterial isolates (B23, B24, B13, B22, P4, B4, and P2) with closely related species based on 16S rRNA gene sequencing is shown in the phylogenetic tree created using the neighbor-joining method (Fig. 2).

Table 2 Accession numbers of isolated bacteria in GenBank database

Screening tests

Corrected aphid mortality rates showed a highly significant difference at 72 h (df = 30, Fr = 3.5, p < 0.0001) under the effect of the 1 × 10⁸ CFU/ml concentration between Bacillus and Pseudomonas characterized isolates, including 5 Pseudomonas (P) and 22 Bacillus (B) isolates compared to controls Bt (Bacillus sp.) HF911367 and Pf (Pseudomonas sp.) HF911366 (Fig. 3).

Fig. 3

Effect of all bacterial isolates against Hyalopterus pruni, 72 h after application at concentration 1 × 10⁸ CFU/ml

Isolate P4 and Bt control strains produced very high mortality levels (95.83 and 93.81%, respectively) significantly different from those of strains B24, B17, B23, B14, P2, and B22 (24% of the total) which have generated fewer high effects on H. pruni mortality, ranging from 71.42 to 85.71%. The control Pseudomonas strain Pf and isolates B4, B13, B2, B15, P3, B16, P1, and B21 (31% of the total), have caused aphid mortalities ranging between 40 and 53%, whereas B18, B9, B20, B5, B19, B7, B8, B1, B3, B6, B12, and B11 strains (41.3% of the total) have induced much low mortality percentages varying from 12 to 34% (Fig. 3). Contrariwise, B10 isolate did not affect aphid mortality.

Evaluation of the selected isolates effect on H. pruni

Global effect with applied concentrations after 24, 48 and 72 h

The estimated mean effect of the 4 applied concentrations between selected isolates (n = 10 isolates) was significantly different (df = 22, Fr = 3.886, P < 0.0001). Aphid H. pruni average mortality observed with all 10 selected isolates (B13, B14, B17, B22, B23, B24, control Bt and P2, P4, P5, control Pf) was less than 20% after 24 h of treatment with the fourth concentrations (C1 to C4). The four isolates (B22, B23, P2 and P4) demonstrated after 72 h greater than 50% mortality rate, but the three isolates (B23, P2 and P4) showed a mortality rate over 50% after 48 h (Fig. 4). At 72 h after treatment, isolate P4 showed the highest cumulative mortality rate (85.81%) on aphid H. pruni, compared to that of P2 and B23 isolates (76.98 to 79.15%), B22 isolate (71.94%) and to that of controls 61.62% (Pf) and 51.02% (Bt). Relatively even at 48 h, P4, P2, and B23 showed high mortality rates of 62.68, 64.15, and 60.20%, respectively, whereas Bt (22%) and Pf (32.39%) controls have induced much low mortality rates (Fig. 4). However, average H. pruni mortality rates were recorded 48.68% (B13), 48.21% (B24), 47.73% (B4), 39.45% (B14), 30.72% (P5), and 30.72% (B17) after 72 h and only counted between 15.84 and 25.60% for B13, B24, and B4, respectively, at 48 h.

Fig. 4

Estimated means mortality of selected isolates 24, 48, 72 h after application at used concentrations

Globally, increasing H. pruni aphid mortality percentages was time dependent (df = 2.000, Fr = 189,669, p < 0.0001). The best mortality rate (56.24%) was recorded after 72 h after treatment, followed by those recorded at 48 h (34.31%), then at 24 h after application of the treatments (9.62% only) (Table 3).

Table 3 Average aphid mortality (%) under selected isolates effect after 24, 48, 72 h of treatment

All the tested bacterial isolates concentrations (C1 to C4) have achieved a similar and low effect (6–14%) at 24 h, and a high mortality rate at the end of 72 h (Fig. 5).

Fig. 5

Aphid H. pruni mortality after 24, 48 and 72 h with each concentration

Concentration C1 induced the highest mortality rate at 48 h (45.63%) and at 72 h (74.11%). Nevertheless, concentrations (C2, C3 and C4) had a convergent effect throughout the treatment period, with no significant difference between C2 (31.91%), C3 (28.92%) and C4 (30.77%) at 48 h, and also between C2 (48.05%), C3 (49.29%) and C3 (53.51%) after 72 h of treatment (Fig. 5). Tukey post hoc comparison showed a significant difference (df = 3, Fr = 16.149, p < 0.0001) between the applied concentrations and within the time of treatment with the concentrations used (df = 6, Fr = 2.529, p = 0.021).

Effect of the concentrations of selected isolates on H. pruni after 72 h of treatment

Accordingly, isolates P4, P5, B24, B17, B23, B14, P2, B22, B4, and B13 (Fig. 3), which have exhibited 50% of aphid mortality and more, were therefore tested at the concentrations (C1 = 1 × 10⁸ UFC/ml, C2 = 1 × 10⁷ UFC/ml, C3 = 1 × 10⁶ UFC/ml, C4 = 1 × 10⁵ UFC/ml), for a duration time of 72 h. Evaluation of pathogenicity at these concentrations showed variable mortality rates on H. pruni (Fig. 6), (df = 33, Fr = 2,368, p = 0.001). At the lowest concentration (C4), bacteria strains B23, B22, B4 and P2 induced high mortalities in H. pruni aphids 96, 91, 85 and 83%, respectively (Fig. 6). Moreover, these mortality rates were higher than those recorded in P4 (65.92%) and B13 (65.61%). With concentrations C2 and C3, P4 isolate caused as well very significant mortality of H. pruni aphids ranging from 80 to 100%, and even rather similar death rates (79 and 73%) with the isolate P2 were detected at these same concentrations. B23 and B22 also recorded high mortality rates at C3 and C2 but were less effective than those of P2 and P4, with rates of 74% (B22), 65% (B23), and 77.42% (B23) at C3, and 50.31% (B22) at C2, where B24 was 50% effective at this concentration. In contrast, isolate B4 recorded high mortality at the lowest concentration while showing low mortality at the other concentrations (C2 and C3). In comparison with the two referenced strains, Bt and Pf, the mortality rate decreased according to the concentrations prepared by Bt, where 45% (C2), 36% (C3), and 24% for the lowest concentration (C4) were recorded. In contrast, the Pf strain gave mortality rates of over 60% at treatments with low concentrations of C3 (73.61%) and C4 (61.80%), with a rate of 55.32% for C2. All isolates tested gave effective mortalities of between 51.37% and 95% at the highest concentration (C1) (Fig. 6).

Fig. 6

Effect of selected isolate at different doses after 72h of application against Hyalopterus pruni

Determination of lethal concentration (LC₅₀, LC₉₀) values

LC₅₀ of isolate P4 (Enteroccocus galinarum) was the lowest (1.08 × 10² CFU/ml) among all the other potentially most efficient isolates after 72 h of treatment and the positive controls Bt and Pf (Table 4). Notably, strain P2 showed the lowest LC90 (1.16 × 10³ CFU/ml) after 72 h of treatment (Table 4).

Table 4 Lethal concentration values (CFU/ml) for B22, B23, P2, P4 isolates compared to Bt (HF911367) Pf (HF911366)

Determination of lethal time (LT₅₀, LT₉₀) values

The lowest LT₅₀ value (33.6 h) for P2 strain effect was recorded after treatment with concentration C4 (1 × 10⁵ CFU/ml). In contrarily, P4 isolate exhibited the lowest LT₉₀ (50.4 h) after treatment with concentration C3 (1 × 10⁶ CFU/ml) (Table 5).

Table 5 Median lethal times values (hours) for B22, B23, P2, P4 isolates compared to Bt (HF911367) and Pf (HF911366)

Discussion

Obtained results have shown that B4, B22, B23, B24, B13, P2, and P4 isolates have generated high mortality rates against H. pruni. These isolates were identified as Bacillus, Pseudomonas, and Enterococci genera, using 16S rRNA gene sequence analysis. Bacillaceae and Pseudomonadaceae were already known to occur in soil (Oulebsir-Mohand kaci et al. 2015a, b). Enterococcaceae have also been isolated from soil (Falcone et al. 2017).

P4 strain (Enteroccocus galinarum) was the most effective to kill H. pruni as the average recorded 85.5% of mortality which was obtained for the four tested concentrations, and still total aphids mortality (100%) has been reached after 72 h with a concentration of 1 × 10⁶ CFU /ml. Several studies have focused on isolating Enterococci species from various gut insects to using these bacteria for biological control, as they cause sepsis and rapid death when trans-locating toward the hemolymph, (Mason et al. 2011). According to the literature, Enterococcus genus has not been tested against aphids (Hemiptera) but in the present study, the species E. gallinarum isolated from soil and tested against H. pruni aphids, has proven an aphicidal effect.

Compared to Pseudomonas, the P2 strain (Pseudomonas sp.) also caused 83% mortality rate after 72 h of treatment with the lowest concentration (C4 = 1 × 10⁵ CFU/ml). P. fluorescens strains have insecticidal activity against agricultural pests, including aphids (Kupferschmied et al. 2013). Our results corroborate those of Paliwal et al (2022), who successfully demonstrated that P. fluorescens presented 100% mortality after 72 h of treatment against the green peach aphid Myzus persicae, but at the concentration of 1 × 10⁷ CFU/ml. Furthermore, Manjula et al (2018) confirmed P. fluorescens efficacy against the cotton aphid, Aphis gossypii, causing 65.3% reduction after 1% foliar application. On the other hand, Hashimoto et al. (2002) identified P. fluorescens and other bacteria as possible pathogens for aphid Myzus persicae.

In this study, the difference in aphid mortalities recorded with P2 isolate (Pseudomonas sp.) in comparison with those of the referenced strain pf (HF911366) may be explained by host insect specificity, given that the referenced strain was tested for its insecticidal effect on L. migratoria larvae and G. mellonella (Oulebsir-Mohand kaci et al. 2015a, b). Thus, Paliwal et al (2022) found different mortality rates having been caused by P. fluorescens (PfR37) on two different species of aphids (Myzus persicae and Aphis fabae).

B. thuringiensis strains B23 and B22 were responsible for the highest H. pruni aphids mortality at the lowest concentration (C4 = 1 × 10⁵ CFU/ml), (96% and 92%, respectively). Our results match with those of Alamuratov et al. (2023) who demonstrated that Bt has an entomopathogenic effect against H. pruni with a 69.5% mortality as the best efficiency obtained at concentration 2 × 10⁸ CFU/ml after 72 h in laboratory conditions. The same authors observed similarly different mortality rates due to other Bt strains using the same concentration. These results are similar with our finding related to variable aphid’s mortalities observed with Bt isolates B13 and B24 that generated low insecticidal potential compared to Bt strains (B23, B22). Previous works also found this difference in insecticidal potential of multiple soil-borne B. thuringiensis strains on lepidopteran and dipteran insects (Astuti et al. 2018) and on the pea aphid Acyrthosiphon pisum (Porcar et al 2009). This difference can be explained by the host specificity of many B. thuringiensis sub-species related to the variation of insecticidal activity of the endotoxins they produce (Yamagiwa et al. 2002).

P4 isolate (Enterococcus gallinarum) was found to be the most potent among the tested isolates. As the Enterococcus’ genus is a component of insects' gut microbiota (Cox and Gilmore 2007) ingesting a small quantity of these bacteria may thus cause intestinal disturbances with commensal bacteria of the same genus, which explains why this low concentration could cause mortality in populations of H. pruni aphids.

In the present study, results highlighted that both strains P4 and P2 displayed the best insecticidal potential against H. pruni aphids. Paliwal et al (2022) mentioned that a low concentration (5.24 × 10¹ CFU/ml) for this same bacteria species was sufficient to cause 50% mortality of several aphid species after 72 h of treatment.

Compared to the other potentially effective strains (P2, P4), Bt strains B22 and B23 showed however high levels of LC and LT against H. pruni aphids. Alamuratov et al. (2023) have found a Bt concentration of 2 × 10⁸ CFU/ml that induced mortality range varying between 63.5 and 35.2% after 72 h of treatment. So, according to our finding, selected Bt soil-borne strains do not have the best insecticidal effects after 72 h of treatment against H. pruni as Pseudomonas and Enterococci strains have. Bt strains used as controls showed lifted LC and LT. This could be due to variations in the insecticidal genes availability, the aphids species utilized, or the applied bioassay methodology and more precisely host specificity (Meddas et al. 2020).

Conclusion

The study involves isolating and characterizing bacteria from cultivated citrus soil, followed by an evaluation of their pathogenic potential against the mealy plum aphid, Hyalopterus pruni. The selected bacterial isolates showed high mortality in H. pruni populations after 72 h of treatment revealed the presence of three different genera of entomopathogenic bacteria. This ensures that citrus soils contain a high diversity of entomopathogenic bacteria. Also, Enterococcus gallinarum species of Enterococci genus was available in cultivated soil ecology and generates the best mortality rate compared to other genera, highlighting the greatest importance of this genus in aphid control applications. Moreover, Bacillus and Pseudomonas genera had a pathogenic effect. The pathogenic effect of a new species, Lysinibacillus fusiformis, of the Bacillus genus, was already known for its insecticidal effect against another order of insects (Lepidoptera). Ultimately, this work will provide an opportunity to develop and study the active molecules and toxins of B. thuringiensis and Pseudomonas sp., which their entomopathogenic effects have already proved and to exploit the impact of the new species, E. gallinarum and L. fusiformis, as microbial bioinsecticides against aphids and various other insect pests.