Biocontrol of Cassida vittata Vill. (Coleoptera: Chrysomelidae) in Sugar Beet Crops Using Streptomyces sp. Strains

Abstract

Cassida vittata Vill. is a major pest of sugar beet crops worldwide. This study evaluated the efficacy of Streptomyces sp. strains E23-2, E23-9, E23-3, and E25-12 in managing this pest under both laboratory bioassays (26 ± 2 °C, 60 ± 10% RH, and 12 h of photoperiod) and field conditions. In the laboratory bioassays, insecticidal and repellent activities were assessed using topical and leaf dip methods. The insecticidal activity test involved five concentrations of bacterial suspensions (10² to 10¹⁰ cfu. mL⁻¹ (Colony-Forming Units per milliliter)) against pest larvae and adults. Only E23-2 and E23-9 strains at concentrations of 10¹⁰ and 10⁸ cfu. mL⁻¹, exhibiting the highest insecticidal activity, were used for the field bioassay. Carbosulfan at 0.25 g/L served as a positive control. Results indicated E23-2’s high efficacy against C. vittata, with the lowest LC₅₀ values: 323.5 (larvae) and 5.1 × 10³ (adults) cfu. mL⁻¹ in topical contact, and 1.9 × 10³ (larvae) and 3.1 × 10⁴ (adults) cfu. mL⁻¹ in the leaf dip method. LT₅₀ values of 3 days for larvae and adults in the topical contact method supported E23-2’s efficacy. E23-2, at 10¹⁰ cfu. mL⁻¹, displayed notable repellency against C. vittata adults (RI = 84.9% at 48 h). In field trials, Henderson–Tilton adjusted rates revealed E23-2’s substantial reductions of 88.6% (larvae) and 85.9% (adults), aligning closely with Carbosulfan’s efficacy. Enzymatic analysis underscored the versatile biocontrol attributes of E23-2, E23-3, and E23-9, providing insights for targeted pest management strategies. Field conditions, notably temperature, can influence the establishment and efficacy of EP bacteria. Further field studies are imperative for a comprehensive understanding of these influencing factors.

1. Introduction

Sugar beet, Beta vulgaris L., is the world’s second most cultivated crop for white sugar production after sugar cane [1]. It contributes to approximately 27% of the world’s total annual sucrose production [2]. Sugar beet has also been developed as an efficient biofuel alternative to fossil fuel energy [3]. Because of its high drought resistance, tolerance to relatively high salt concentrations, and adaptability to various ecological conditions [4], sugar beet cultivation in Morocco has significantly expanded over the past decade, with production increasing from 468,000 tons in 2008 to 591,000 tons in 2019, accompanied by a substantial improvement in yield.

Sugar beet crops are known to be susceptible to a range of insect pests, with one of the most notable being the tortoise beetle, Cassida vittata Vill. (Coleoptera: Chrysomelidae) [5,6]. Both the larval and adult stages of C. vittata feed on sugar beet leaves, leading to crop losses due to leaf damage, defoliation, and a subsequent reduction in the sugar content of the affected plants [7]. Severe infestations of sugar beet with C. vittata have resulted in a significant reduction of 40.10% in root weight and 56.20% in sugar content [8].

Cassida vittata is widespread in North Africa and Europe, causing significant damage in countries like Italy, Spain, Greece, Turkey, Algeria, Egypt, and Morocco [9]. This pest is mainly controlled by conventional chemical insecticides [4], which can have harmful effects on human and animal health and the environment and, in some cases, limit international trade in sugar beet seeds and plants among the countries interested in this crop cultivation [10]. Therefore, to reduce insecticide use, many alternative management strategies have been explored in many countries, such as the use of mineral oils, plant extracts, entomopathogenic microorganisms, and biological control agents (predators and parasitoids) [4,10]. The sugar beet fields have several natural predators, parasitoids, and entomopathogenic factors that should be conserved to maintain the natural balance in the field [10]. Entomopathogenic nematodes (EPNs) from the genera Steinernema and Heterorhabditis, such as H. bacteriophora and S. feltiae, are known for their effectiveness in controlling various insect pests on economic crops [11]. These nematodes have demonstrated their ability to cause mortality in C. vittata larvae, pupae, and adults under field conditions in Egypt [4]. Within alternative management approaches, entomopathogenic microorganisms have been known for a long time to be an important factor in the natural mortality of insects [12].

Bazazo et al. [10] recorded mortality rates ranging from 20.0 to 45.0% for C. vittata larvae and 10.0 to 40.0% for adults in laboratory tests, while field tests showed larval reductions of 12.86 to 43.99% and adult reductions of 14.1 to 39.8% due to Bacillus aryabhattai exposure. Bacillus species exhibit the production of crystalline proteins during their stationary growth phase, effectively delivering lethal effects on coleopterous insects [13]. Several bacterial species, including Streptomyces sp., Pseudomonas entomophila, Xenorhabdus sp., Burkholderia sp., Yersinia entomophaga, Chromobacterium sp., Saccharopolyspora sp., and Bacillus sp., have garnered attention in the commercial production of biopesticides [14,15]. Pseudomonas-derived biopesticides have demonstrated effectiveness in controlling key agricultural pests such as the two-spotted spider mite (Tetranychus urticae Koch (Acari: Tetranychidae)) [16] and the cochineal Dactylopius opuntiae (Cockerell) (Hemiptera: Dactylopiidae) [17].

Pseudomonas fluorescens chitinases effectively degrade parasites by breaking down their chitinous exoskeletons [18]. The fungal and bacterial chitinase enzymes have been employed to suppress larvae of the bollworm (Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)) and stem borer species, including Sesamia calamistis (Hampson) (Lepidoptera: Noctuidae), Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae), and Eldana saccharina (Walker) (Lepidoptera: Pyralidae), by disabling the chitin found in the peritrophic membrane [19,20]. Furthermore, bacteria capable of producing chitinase and protease enzymes with activity similar to chemical compounds are considered promising in the field [17]. Numerous studies have underscored the pivotal role of specific secondary metabolites produced by Streptomyces species in the management of agriculturally significant pests, including Plutella xylostella (Linnaeus) (Lepidoptera: Yponomeutidae) [21], Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) [22], Spodoptera littoralis (Biosduval) (Lepidoptera: Noctuidae) [23], Anopheles mosquito larvae (Theobald) (Diptera: Culicidae) [24], Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) [25], Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) [26], Culex quinquefasciatus Say (Diptera: Culicidae) [27], and Musca domestica (Li) (Diptera: Muscidae) [28]. More recently, the ethyl acetate (EA) extract of Streptomyces sp. KSF103 has shown potential for eco-friendly mosquito control, targeting Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse), Anopheles cracens Sallum and Peyton, and Culex quinquefasciatus Say (Diptera: Culicidae) [29]. The protein extract from Streptomyces sp. SP5 exhibited significant larvicidal activity against the melon fruit fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), with an LC₅₀ of 308.92 μg/mL [30]. Streptomyces sp. SA61 demonstrated high insecticidal activity against Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae) with an LC₅₀ of 6.949 mg/L at 72 h [31]. Additionally, Streptomyces species produce various secondary metabolites with fungicidal and bactericidal properties that help control plant diseases and promote environmental safety [32]. Streptomyces plumbeus produces polyene compounds effective against Botrytis cinerea Pers. (Helotiales: Sclerotiniaceae) [33]. Kasugamycin, from Streptomyces kasugaensis, controls rice blast (Pyricularia oryzae Cavara (Magnaporthales: Magnaporthaceae)) and Pseudomonas diseases by inhibiting protein biosynthesis [34]. Furthermore, Streptomyces termitum ATC-2 produces aloesaponarin II, which is effective against rice bacterial blight [35]. Streptomyces sp. RM365 suppresses Xanthomonas campestris pv. glycines on soybeans, and strain OE7 inhibits Pectobacterium species on potatoes [36].

Insects are mainly infected by bacteria through ingestion, with potential routes via the egg, integument, and trachea. Pathogenic bacteria in the digestive tract can produce harmful enzymes, causing potential harm or death [37]. Numerous native Streptomyces strains from Morocco were identified [38]. Yet, their efficacy against C. vittata has not been tested. This study evaluates the effectiveness of five indigenous Streptomyces strains in controlling C. vittata larvae and adults under laboratory and field conditions.

2. Materials and Methods

2.1. The Insect Rearing

Sugar beet flea beetles, C. vittata, were collected from infested sugar beet fields in the Zemamra locality, Casablanca-Settat region, Morocco (33°15′ N, 8°30′ W, Elevation 165 m). Field surveys were conducted at 15-day intervals between 15 April 2023, and 15 June 2023, totaling five surveys. Approximately 400 reproductively active male and female C. vittata were used to establish laboratory colonies.

These beetles were reared in entomological cages (80 × 80 × 80 cm), featuring wooden frames covered with mesh fabric for proper ventilation. They were provided with one-month-old B. vulgaris leaves, which were replaced with new ones every two weeks to ensure a continuous supply of food and oviposition support to the adults. The B. vulgaris leaves were collected from sugar beet fields in the Zemamra locality. The beetle population was maintained at a temperature of 25 ± 1 °C, with relative humidity ranging from 65 to 80%, and subjected to a photoperiod of 18:8 (L:D) hours. Before conducting laboratory experiments, the sugar beet flea beetle was reared for at least one generation on B. vulgaris. To bolster the insect population and determine their age, 24 h old first instar larvae of C. vittata were transferred to a separate cage with conditions as described above to complete their development. Voucher specimens of C. vittata, confirmed by the authors, have been archived at the Insectarium of the National Institute of Agricultural Research in Zemamra, Morocco.

2.2. Entomopathogenic Bacteria

Five strains of Streptomyces sp. were examined for their efficacy against C. vittata, following their isolation from Moroccan soil [38]. The strains include Streptomyces bellus E23-2 (NCBI GenBank Acc. No: OM883988), Streptomyces galilaeus E23-9 (NCBI GenBank Acc. No: OM883992), Streptomyces africanus E23-3 (NCBI GenBank Acc. No: OM883989), and Streptomyces bellus E25-12 (NCBI GenBank Acc. No: OM883998). After revival from glycerol stocks, the strains were cultured on ISP-2 medium in Petri dishes (14.5 cm in diameter) using the striation method as described by Rossi-Tamisier et al. [39]. The Petri dishes were incubated at 28 °C, and the cultures were monitored daily over a period of 10 days for any signs of contamination, such as abnormal growth or colony morphology. Additionally, we performed Gram staining and examined the pure strains under an optical microscope (Olympus CX43RF, Olympus Corporation, Tokyo, Japan) to confirm their purity. Any cultures exhibiting contamination were discarded, and new subcultures were prepared to maintain strain purity. The physiological and biochemical traits of the Streptomyces sp. strains were assessed based on established methodologies [38,40]. These included the production of melanoid pigments, tolerance to varying concentrations of sodium chloride (NaCl) and pH values, growth at different temperature levels, and assimilation of carbohydrates [38]. The tested strains have previously demonstrated efficacy in laboratory and field trials for controlling the cactus cochineal D. opuntiae [41].

2.3. Laboratory Trials

The pathogenicity of the five Streptomyces sp. strains was investigated against C. vittata larvae and adults under laboratory conditions (26 ± 2 °C, 60 ± 10% RH, and 12 h of photoperiod). The assessment was conducted using both topical and leaf dip bioassays. The strains were tested at various concentrations (10², 10⁴, 10⁶, 10⁸, and 10¹⁰ cfu. mL⁻¹). Each strain was cultured in 100 mL of nutrient broth with 0.1% Tween 20 and incubated in an orbital shaker incubator at 28 °C and 150 rpm for 24 h. Following incubation, a centrifugation step at 10,000 rpm was carried out, and bacterial cell concentrations were measured at 640 nm using a spectrophotometer (Optizen 3220UV/VIS double beam, Mecasys, South Korea) [42]. Tap water was utilized as a negative control, while Marshal 20% EC (250 g of Carbosulfan per liter, applied at 0.25 g/L; Delta Chemical Co., Paris, France), diluted in tap water, was used as a positive control. This chemical insecticide served as a positive control treatment because of its known toxicity against different stages of C. vittata. Carbosulfan at 0.25 g/L effectively reduced C. vittata larvae by 90.7% and 93.4% one day after treatment, reaching 98.5% and 99.78% reductions ten days later in the first and second seasons, respectively [43]. Profenofos and carbosulfan are efficient compounds against all stages of the tortoise beetle [44].

2.3.1. Topical Contact Bioassays

Ten-day-old (since eclosion) adults (Trial 1) and 10-day-old (since hatching) larvae (Trial 2) were placed in groups of ten in 14.5 cm diameter Petri dishes supplied with one-month-old B. vulgaris leaves. In both trials, 2 mL of each treatment were applied via an A Potter spray tower (Burkard Scientific Ltd., Uxbridge, UK) as a mist over the larvae, adults, and host plants at 150 kPa, resulting in a spray deposit of 0.0015 mL/cm² [41]. After treatment, the insects were transferred to plastic containers (15 cm in length, 10 cm in width, 5 cm in height) containing damp filter paper and untreated young tender leaves of B. vulgaris. Plant materials were replaced daily over an 8-day period. These plastic containers (10 individuals each) were arranged in a completely randomized design (CRD) with four replications. The numbers of alive and dead insects were recorded at 1, 3, and 8 days after application, using a binocular loupe (SFC-11, MOTIC^®, Motic Instruments, Richmond, British Columbia, Canada). Adults were classified as “alive” if their mouthparts and antennae moved when touched with a fine paintbrush, and larvae were considered alive when they appeared green (as opposed to greenish–brown, which indicated they were dead). To ensure the reproducibility of results, all experiments were independently repeated thrice over time, resulting in a total of 120 larvae and 120 adults treated with each treatment.

2.3.2. Leaf Dip Bioassays

Beta vulgaris young tender leaves (about 500 g) were dipped and gently agitated in tap water (negative control) or the insecticide solution (positive control) of an application dose (similar to topical bioassay) or EP bacteria solution for 5 s and then air-dried for 5–10 min before being placed into plastic containers (15 cm in length, 10 cm in width, 5 cm in height). Groups of ten larvae (Trial 1) and adults (Trial 2) collected from the laboratory colony were introduced into the plastic containers, and mortality was assessed at 1, 3, and 8 days after introduction using the criteria described above. The experimental design and study replications were the same as those used for the topical treatment of larvae and adults; thus, a total of 120 larvae and 120 adults were treated with each treatment.

2.3.3. Repellent Activity

This experiment aimed to assess the repellent effectiveness of the four Streptomyces sp. strains tested on C. vittata adults. The concentration of 10¹⁰ cfu∙ mL⁻¹, causing maximum mortality in C. vittata larvae and adults, was employed. Repellent activity was evaluated using the modified choice test by Pascual-villalobos and Robledo [45]. The experimental setup involved two plastic containers (15 cm in length, 10 cm in width, 5 cm in height), each containing either control or treated B. vulgaris young leaves, connected by a 10 cm long translucent hose (1 cm diameter) with a central hole for introducing the tortoise beetles. Beta vulgaris young leaves were immersed for 20 s in either a suspension of Streptomyces sp. strain (10¹⁰ cfu ∙ mL^–1) or tap water, placed under laboratory conditions until dry, and then positioned in the corresponding container within the repellent apparatus. Twenty C. vittata adults (with a male-to-female sex ratio of 1:1) were then gently transferred through the hole in the middle of the linked hose and sealed. This design allowed the beetles to move freely between the control and treated boxes. The experimental layout comprised a 4 Streptomyces sp. strains at 10¹⁰ cfu ∙ mL⁻¹ and control (tap water) × 3 (time intervals) factorial, utilizing a randomized complete block design with four replications, and all experiments were repeated three times. Experiments were conducted in a growth room maintained at 26 ± 2 °C, with a relative humidity of 60 ± 10%, and a photoperiod of 12:12 h light–dark cycle. The migration of beetles to one of the two linked containers was determined at 12, 24, and 48 h after release. The repellency index was calculated using the formula established by Pascual-villalobos and Robledo [45]:

$$\mathrm{R}\mathrm{I}={\displaystyle \frac{\mathrm{C}-\mathrm{T}}{\mathrm{C}+\mathrm{T}}}\times 100$$

where RI—repellency index, C—number of C. vittata adults in the control plastic containers, and T—number of C. vittata adults in the treated plastic containers.

2.4. Field Trials

The study was conducted at the experimental field station of the Doukkala Regional Office for Agricultural Development, located in the Zemamra locality, Morocco (32°15′ to 33°15′ N, 7°55′ to 9°15′ W, and an elevation of 165 m) during the 2022–2023 agricultural campaign. The Zemamra zone is situated between El Jadida and Safi Provinces and is situated in a semi-arid ecological zone where the average annual rainfall is 330 mm (ranging from 113 to 607 mm), and temperatures vary from −1 °C (minimum) in December and January to 45 °C (maximum) in July and August. The daily temperature during this study ranged from 8 to 28 °C and was recorded using an iMetos electronic weather station (iMetos AG/CP/DD 280, Pessl Instruments GmbH, Weiz, Austria).

The soils in the Zemamra zone are characterized by the predominance of Tirs soil (82%), followed by Rmels soil (12%), and Hamri soil (5%). This experiment was carried out in cultivated B. vulgaris plots with a plant density of 10 plants/m². Each plot comprised 100 plants, equivalent to an area of 10 m², with adjacent plots separated by rows of 100 plants, approximately 9 m apart. For field tests, Streptomyces sp. strains E23-2 and E23-9, at concentrations of 10¹⁰ and 10⁸ cfu. mL⁻¹, and Carbosulfan applied at a concentration of 0.25 g/L, which showed the highest mortality against both larvae and adults of C. vittata in laboratory bioassays, were selected. Three replications of each of the six treatments (two EP-bacteria treatments at two doses, chemical insecticide, plus untreated control) were arranged in a randomized complete block design [46].

The test solutions were applied using a laboratory sprayer (Burkard Scientific Ltd., Uxbridge, UK) to ensure complete coverage. Twenty whole plants, including their roots, were manually collected from each plot and placed in a cardboard box 1 day before treatment and at 1, 3, 6, and 12 days after treatment (DAT). These plants were then examined in the laboratory, and the numbers of tortoise beetles on each plant were counted under stereomicroscopes. This experiment was repeated twice over different times. The rate of population reduction at each sampling time was corrected using the Henderson–Tilton formula [47]:

$$\mathrm{R}\left(\mathrm{\%}\right)=1-{\displaystyle \frac{\mathrm{T}2\times \mathrm{C}1}{\mathrm{T}1\times \mathrm{C}2}}\times 100$$

where T1 and T2 represent the counts of surviving insects in a treated plot before the treatment and at a specific time point following the treatment, respectively. Conversely, C1 and C2 denote the counts of living insects in an untreated control plot before the treatment and at the same specific time point following the treatment.

2.5. Mechanism of Action of Streptomyces sp. Strains

2.5.1. Chitinase Production

The capacity of the two prominent Streptomyces sp. strains, E23-2 and E23-9, which exhibited notable efficacy against both C. vittata larvae and adults, to produce chitinase was assessed employing the method reported by Cattelan et al. [48]. Each strain was cultured on chitin medium and subjected to a 72 h incubation at 28 °C. The presence of a clear zone surrounding the colony served as an indicator of chitin solubilization by chitinase-producing bacteria.

2.5.2. Cellulase Production

The qualitative assessment of cellulase production by the two strains was conducted using M9 medium agar supplemented with 1.2 g LG1 yeast extract and 10 g LG1 cellulose, following the procedure detailed by Miller et al. [49]. A distinct halo observed around the bacterial colony after an 8-day incubation at 28 °C was considered indicative of successful cellulase production.

2.5.3. Protease Production

The evaluation of protease production by the two strains was performed on skim milk agar, in accordance with the method described by Jha et al. [50]. The strains were inoculated onto the medium and allowed to incubate for 48 h at 28 °C. The presence of a light area around the colony was interpreted as evidence of protease production.

2.6. Data Analysis

Mortality data in lab bioassays were corrected with Abbott’s formula [51] and statistically analyzed using ANOVA. Mean differences were assessed with Tukey’s LSD test at a 0.05 significance level. Probit analysis in IBM SPSS 23.0 software was adopted to determine LC₅₀ values for different treatments by transforming mortality data into probits and concentrations into Probit log10 (dose). Predicted LC₅₀ values were obtained from probit lines before analysis. The Finney method was used to determine the lethal time (LT₅₀) in probit analysis [52]. The calculation of lethal concentration (LC) and its 95% confidence limits (CL) relied on precise estimation of log (CL) variances [53]. The Kaplan–Meier survival analysis was used to describe the median lethal time (LT₅₀) and mean survival time for each treatment using SPSS 23.0. One-way ANOVA tests were used to examine differences in doses and exposure times with significance levels set at p < 0.05, and significant distinctions between variables were confirmed using Tukey’s LSD test. In the field experiment, the counts of surviving larvae and adults, as well as population reduction rates for different treatments, were analyzed using ANOVA with a randomized complete block design (RCBD). Tukey’s LSD test was employed to differentiate means. SPSS 23.0 software was used for these analyses, with treatment as a fixed effect and replication as a random factor.

3. Results

3.1. Biochemical and Physiological Characteristics of Streptomyces sp. Strains

The growth, biochemical, and physiological characteristics of the four Streptomyces sp. strains tested are presented in

Table 1. Biochemical and physiological characteristics of Streptomyces sp. strains.

Characteristics	Streptomyces sp. Strains
	E23-2	E23-3	E23-9	E25-12
Assimilation Ribose	-	+	-	+
Melezitose	+	3+	-	-
Manitol	3+	3+	-	3+
Trehalose	3+	3+	3+	3+
Cellobiose	3+	3+	3+	3+
Sucrose	3+	3+	3+	3+
Raffinose	3+	3+	3+	3+
Xylose	3+	3+	+	3+
Melibiose	3+	3+	-	3+
Mannose	3+	3+	3+	3+
Fructose	3+	2+	3+	3+
Galactose	3+	3+	3+	3+
Maltose	3+	2+	2+	3+
Glucose	3+	3+	3+	3+
pH tolerance 4.63	-	-	-	-
5.33	2+	+	+	+
6.41	2+	3+	2+	2+
7.31	3+	3+	3+	3+
8.28	3+	3+	3+	3+
9.27	3+	2+	3+	3+
10.03	2+	2+	+	3+
NaCl tolerance 1%	3+	3+	3+	3+
2%	3+	3+	2+	3+
3%	3+	3+	+	3+
4%	3+	3+	+	2+
5%	2+	3+	+	2+
7%	2+	+	-	+
10%	-	-	-	-
Growth on 4 °C	-	-	-	-
28 °C	3+	3+	3+	3+
37 °C	3+	3+	2+	2+
46 °C	-	-	-	-

(-) no growth; (+) low growth; (2+) intermediate growth; (3+) good growth.

. The results show that all the strains have good growth on various carbon sources such as trehalose, cellobiose, sucrose, raffinose, xylose, melibiose, mannose, fructose, galactose, maltose, and glucose. In terms of pH tolerance, E23-3 and E25-12 show better tolerance at higher pH levels compared with E23-2 and E23-9. Similarly, all the strains show good tolerance to NaCl up to 3%, with decreasing tolerance at higher concentrations (none of the strains can grow at 10% NaCl). Growth at different temperatures indicates that all strains can grow at 28 °C and 37 °C, while none of the strains can grow at 4 °C and 46 °C.

3.2. Laboratory Trials

The effects of different concentrations of the tested strains on the percentage mortality of C. vittata larvae and adults in lab bioassays are shown in

Table 2. Effects of different concentrations of Streptomyces sp. strains on the percentage mortality of Cassida vittata, larvae.

Lab Bioassays	Time (Days)	Concentrations (CFU mL-1)	Streptomyces sp. Strains				Carbosulfan at 0.25 g/L
			E23-2	E23-9	E23-3	E25-12
Topical contact	1	102	25.0 ± 5.8 Cb	22.5 ± 3.8 Db	17.5 ± 5.0 Dbc	13.3 ± 4.4 Dc	60.0 ± 6.7 a
		104	34.2 ± 4.5 Cb	26.7 ± 5.6 Dbc	21.7 ± 4.2 Dcd	15.0 ± 5.0 Dd	60.0 ± 6.7 a
		106	50.0 ± 10.0 Bab	40.0 ± 5.0 Cbc	35.0 ± 7.5 Ccd	27.5 ± 6.7 Cd	60.0 ± 6.7 a
		108	57.5 ± 9.2 ABab	51.7 ± 7.2 Bab	47.5 ± 6.3 Bbc	38.3 ± 7.2 Bc	60.0 ± 6.7 a
		1010	68.3 ± 7.5 Aa	64.2 ± 8.5 Aab	60.8 ± 9.3 Aab	50.8 ± 9.3 Ab	60.0 ± 6.7 a
	3	102	35.0 ± 7.5 Db	29.2 ± 3.1 Cbc	25.8 ± 6.5 Cbc	24.2 ± 9.2 Cc	85.8 ± 5.6 a
		104	50.0 ± 3.3 Cb	45.8 ± 5.8 Bbc	39.2 ± 6.1 Bcd	36.7 ± 8.3 BCd	85.8 ± 5.6 a
		106	64.2 ± 7.8 Bb	51.7 ± 8.6 Bc	49.2 ± 10.9 Bc	41.7 ± 8.6 Bc	85.8 ± 5.6 a
		108	83.3 ± 6.7 Aa	70.8 ± 6.3 Ab	65.8 ± 8.2 Ab	60.0 ± 10.0 Ab	85.8 ± 5.6 a
		1010	89.2 ± 6.3 Aab	79.2 ± 3.1 Aab	76.7 ± 7.8 Abc	66.7 ± 11.1 Ac	85.8 ± 5.6 a
	8	102	45.8 ± 9.2 Db	40.0 ± 3.3 Dbc	33.3 ± 5.6 Dcd	27.5 ± 6.3 Cd	91.7 ± 5.8 a
		104	59.2 ± 3.1 Cb	52.5 ± 7.5 Cbc	47.5 ± 6.3 Ccd	39.2 ± 4.6 BCd	91.7 ± 5.8 a
		106	76.7 ± 5.6 Bb	65.0 ± 6.7 Bc	56.7 ± 6.7 Bcd	50.0 ± 8.3 Bd	91.7 ± 5.8 a
		108	89.2 ± 3.1 Aab	82.5 ± 10.4 Aabc	77.5 ± 9.9 Abc	73.3 ± 13.9 Ac	91.7 ± 5.8 a
		1010	90.0 ± 3.3 Aa	89.2 ± 3.1 Aa	85.0 ± 5.8 Aa	86.7 ± 5.6 Aa	91.7 ± 5.8 a
Leaf dip	1	102	19.2 ± 7.6 Cb	16.7 ± 6.7 Cb	11.7 ± 2.8 Dbc	7.5 ± 3.8 Cc	54.2 ± 8.2 a
		104	28.3 ± 6.9 Cb	20.8 ± 6.1 Cbc	15.8 ± 4.9 Dcd	9.2 ± 6.1 Cd	54.2 ± 8.2 a
		106	44.2 ± 9.9 Bab	34.2 ± 7.5 Bbc	29.2 ± 11.0 Ccd	21.6 ± 5.6 Bd	54.2 ± 8.2 a
		108	51.7 ± 11.9 ABa	45.8 ± 10.8 Bab	41.7 ± 10.3 Bab	32.5 ± 10.0 Bb	54.2 ± 8.2 a
		1010	62.5 ± 10.0 Aab	60.0 ± 11.7 Aab	55.0 ± 10.8 Aab	45.0 ± 12.5 Ab	54.2 ± 8.2 a
	3	102	29.2 ± 9.3 Db	23.3 ± 5.6 Cbc	20.0 ± 6.7 Cbc	18.3 ± 6.9 Cc	80.0 ± 5.0 a
		104	43.3 ± 6.7 Cb	40.0 ± 6.7 Bbc	33.3 ± 8.9 BCbc	30.8 ± 10.8 BCc	80.0 ± 5.0 a
		106	58.3 ± 8.9 Bb	45.8 ± 10.1 Bbc	44.2 ± 15.8 Bbc	35.8 ± 11.5 Bc	80.0 ± 5.0 a
		108	77.5 ± 5.8 Aa	65.8 ± 10.1 Ab	69.0 ± 8.3 Ab	54.2 ± 13.2 Ab	80.0 ± 5.0 a
		1010	83.3 ± 8.9 Aa	73.3 ± 5.6 Aab	71.7 ± 8.3 Aab	60.8 ± 14.4 Ab	80.0 ± 5.0 a
	8	102	40.0 ± 10.0 Db	34.2 ± 5.8 Cbc	27.5 ± 5.4 Ccd	21.7 ± 8.6 Cd	83.3 ± 11.1 a
		104	53.3 ± 5.6 Cb	46.7 ± 10.6 BCb	41.7 ± 10.0 Bbc	33.3 ± 7.2 BCc	83.3 ± 11.1 a
		106	70.8 ± 6.1 Bab	59.2 ± 9.3 Bbc	50.8 ± 4.7 Bcd	44.2 ± 12.2 Bd	83.3 ± 11.1 a
		108	83.3 ± 7.2 Aa	76.7 ± 12.2 Aa	71.7 ± 10.0 Aa	67.5 ± 17.5 Aa	83.3 ± 11.1 a
		1010	84.2 ± 5.8 Aa	83.3 ± 7.2 Aa	79.2 ± 3.1 Aa	80.8 ± 7.8 Aa	83.3 ± 11.1 a

Within columns means followed by the same capital letters are not statistically different according to Tukey’s LSD test at α = 0.05. Within lines means followed by the same lower-case letters are not statistically different according to Tukey’s LSD test at α = 0.05.

and

Table 3. Effects of different concentrations of Streptomyces sp. strains on the percentage mortality of Cassida vittata, adults.

Lab Bioassays	Time (Days)	Concentrations (CFU mL−1)	Streptomyces sp. Strains				Carbosulfan at 0.25 g/L
			E23-2	E23-9	E23-3	E25-12
Topical contact	1	102	15.8 ± 5.8 Cb	14.2 ± 4.9 Cb	9.2 ± 3.1 Dbc	4.2 ± 5.6 Cc	55.8 ± 8.2 a
		104	25.8 ± 6.5 Cb	19.2 ± 4.6 Cbc	10.8 ± 1.5 Dcd	5.8 ± 5.8 Cd	55.8 ± 8.2 a
		106	40.8 ± 7.8 Bb	34.2 ± 9.2 Bbc	25.0 ± 6.7 Ccd	15.8 ± 4.9 Bd	55.8 ± 8.2 a
		108	53.3 ± 8.9 Aab	42.5 ± 6.7 Bbc	37.5 ± 6.3 Bcd	28.3 ± 7.2 Ad	55.8 ± 8.2 a
		1010	60.8 ± 6.1 Aa	56.7 ± 13.9 Aa	50.8 ± 9.3 Aa	36.7 ± 10.0 Ab	55.8 ± 8.2 a
	3	102	25.8 ± 6.5 Db	20.8 ± 4.6 Cb	19.2 ± 3.1 Dbc	11.7 ± 5.6 Dc	75.8 ± 7.9 a
		104	42.5 ± 9.2 Cb	37.5 ± 6.3 Bb	31.7 ± 8.6 Cbc	22.5 ± 6.3 Cc	75.8 ± 7.9 a
		106	56.7 ± 7.2 Bb	46.7 ± 10.6 Bbc	38.3 ± 11.4 Ccd	30.0 ± 6.7 Cd	75.8 ± 7.9 a
		108	74.2 ± 8.4 Aa	60.0 ± 5.0 Ab	53.3 ± 6.1 Bb	41.7 ± 5.6 Bc	75.8 ± 7.9 a
		1010	82.5 ± 7.5 Aab	68.3 ± 6.9 Abc	64.2 ± 6.5 Acd	53.3 ± 8.3 Ad	75.8 ± 7.9 a
	8	102	35.0 ± 9.2 Db	33.3 ± 9.4 Cb	27.5 ± 6.3 Cbc	19.2 ± 4.6 Ec	88.3 ± 7.2 a
		104	52.5 ± 10.4 Cb	45.0 ± 8.3 Bbc	38.3 ± 5.8 Bcd	28.3 ± 5.6 Dd	88.3 ± 7.2 a
		106	68.3 ± 5.8 Bb	55.8 ± 5.8 Bc	47.5 ± 6.7 Bcd	38.3 ± 5.6 Cd	88.3 ± 7.2 a
		108	85.8 ± 8.9 Aab	74.2 ± 10.8 Abc	65.8 ± 7.5 Acd	55.0 ± 7.5 Bd	88.3 ± 7.2 a
		1010	88.3 ± 5.6 Aa	82.5 ± 6.3 Aa	73.3 ± 6.7 Ab	66.7 ± 4.4 Ab	88.3 ± 7.2 a
Leaf dip	1	102	10.0 ± 3.3 Cb	8.3 ± 6.9 Cb	3.3 ± 5.0 Db	1.7 ± 2.8 Bb	50.0 ± 10.0 a
		104	20.0 ± 10.0 Cb	13.3 ± 7.2 Cbc	5.0 ± 5.0 Dcd	2.5 ± 3.8 Bd	50.0 ± 10.0 a
		106	35.0 ± 10.0 Bb	28.3 ± 11.7 Bbc	19.2 ± 9.3 Ccd	10.0 ± 6.7 Bd	50.0 ± 10.0 a
		108	47.5 ± 12.1 ABa	36.7 ± 8.3 ABab	31.7 ± 8.6 Bbc	22.5 ± 8.8 Ac	50.0 ± 10.0 a
		1010	55.0 ± 8.3 Aa	50.8 ± 16.0 Aa	45.0 ± 11.7 Aab	30.8 ± 12.6 Ab	50.0 ± 10.0 a
	3	102	20.0 ± 10.0 Cb	15.0 ± 7.5 Dbc	13.3 ± 4.4 Cbc	5.8 ± 5.8 Dc	67.5 ± 11.7 a
		104	36.7 ± 12.2 Bb	31.7 ± 7.2 Cb	25.8 ± 10.8 BCbc	16.7 ± 7.8 CDc	67.5 ± 11.7 a
		106	50.8 ± 7.6 Bb	40.8 ± 14.3 BCbc	32.5 ± 14.6 Bcd	24.2 ± 10.8 BCd	67.5 ± 11.7 a
		108	68.3 ± 10.6 Aab	54.2 ± 7.5 ABbc	47.5 ± 9.2 Acd	35.8 ± 6.5 ABd	67.5 ± 11.7 a
		1010	76.7 ± 10.0 Aab	62.5 ± 9.6 Ab	58.3 ± 6.9 Abc	47.5 ± 11.3 Ac	67.5 ± 11.7 a
	8	102	29.2 ± 9.3 Db	27.5 ± 9.6 Cb	22.5 ± 7.5 Cbc	13.3 ± 5.6 Dc	80.8 ± 10.8 a
		104	47.5 ± 12.9 Cb	40.0 ± 11.7 BCb	33.3 ± 9.4 BCbc	22.5 ± 7.5 Dc	80.8 ± 10.8 a
		106	62.5 ± 7.5 Bb	51.7 ± 7.2 Bbc	41.7 ± 8.3 Bcd	33.3 ± 8.3 Cd	80.8 ± 10.8 a
		108	79.2 ± 11.1 Aa	69.2 ± 11.0 Aab	60.8 ± 9.3 Abc	50.0 ± 8.3 Bc	80.8 ± 10.8 a
		1010	81.7 ± 5.6 Aa	77.5 ± 6.7 Aab	68.3 ± 7.2 Abc	61.7 ± 5.6 Ac	80.8 ± 10.8 a

Within columns means followed by the same capital letters are not statistically different according to Tukey’s LSD test at α = 0.05. Within lines means followed by the same lower-case letters are not statistically different according to Tukey’s LSD test at α = 0.05.

, respectively. The experiments were conducted using two application methods (topical contact and leaf dip). Mortalities rose with increasing Streptomyces sp. strain concentrations in both methods for all tested strains. Mortality also increased with time, with higher mortality observed on later days (8 DAT). The 1-, 3-, and 8-day larvae and adults’ mortality in the untreated check was 0%. For larvae, in the topical contact method, the greatest 1-day larva mortality was achieved by E23-2 applied at 10⁶, 10⁸, and 10¹⁰ cfu. mL⁻¹, E23-9 applied at 10⁸ and 10¹⁰ cfu. mL⁻¹, E23-3 applied at 10¹⁰ cfu. mL⁻¹, and Carbosulfan applied at 0.25 g/L. Three days after treatment, the greatest larva mortality was achieved by E23-2 applied at 10⁸ and 10¹⁰ cfu. mL⁻¹, E23-9 applied at 10¹⁰ cfu. mL⁻¹, and Carbosulfan applied at 0.25 g/L. The greatest 8-day larva cumulative mortality was achieved by Carbosulfan at 0.25 g/L (91.7%), E23-2 at 10¹⁰ (90.0%) and 10⁸ (89.2%) cfu. mL⁻¹, E23-9 at 10¹⁰ (89.2%) and 10⁸ (82.5%) cfu. mL⁻¹, E23-3 at 10¹⁰ cfu. mL⁻¹ (85.0%), and E25-12 at 10¹⁰ cfu. mL⁻¹ (86.7%). At the end of the experiment, larva cumulative mortality for E23-2 was at 10⁶ cfu. mL⁻¹, E23-3, and E25-12 at 10⁸ cfu. mL⁻¹ reached 76.7, 77.5, and 73.3% mortality, respectively. The lowest percentage of larva mortality was achieved at 8 days after treatment with the lowest rate of E25-12 at 10² cfu. mL⁻¹. In the leaf dip method, similar trends were observed. One day after introduction, the greatest larva mortality was achieved by E23-2 applied at 10⁶, 10⁸, and 10¹⁰ cfu. mL⁻¹, E23-9 and E23-3 applied at 10⁸ and 10¹⁰ cfu. mL⁻¹, and Carbosulfan applied at 0.25 g/L. Three days after introduction, the greatest mortality was observed on plants treated with E23-2 at 10⁸ and 10¹⁰ cfu. mL⁻¹, E23-9 and E23-3 at 10¹⁰ cfu. mL⁻¹, and Carbosulfan applied at 0.25 g/L. Eight days after introduction, there were no differences among Carbosulfan applied at 0.25 g/L, E23-2, E23-3, E23-9, and E25-12 at 10⁸ and 10¹⁰ cfu. mL⁻¹, and E23-2 at 10⁶ cfu. mL⁻¹, but they still achieved significantly greater mortality than the other treatments. E25-12 at 10² cfu. mL⁻¹ was the least toxic to larvae (21.7% mortality at the end of the experiment).

At 1 day after treatment, the greatest adult mortality in the topical contact method was achieved by Carbosulfan applied at 0.25 g/L, E23-2 applied at 10⁸, and 10¹⁰ cfu. mL⁻¹, and E23-9 and E23-3 applied at 10¹⁰ cfu. mL⁻¹. Three days post-treatment, the highest adult mortality was achieved by E23-2 at 10⁸ and 10¹⁰ cfu. mL⁻¹ and Carbosulfan applied at 0.25 g/L, reaching 74.2, 82.5, and 75.8%, respectively. After 8 days post-treatment, E23-2 at 10⁸ and 10¹⁰ cfu. mL⁻¹ and Carbosulfan applied at 0.25 g/L reached 85.8, 88.3, and 88.3% mortality, respectively. Eight DAT, there were no significant differences in the percent mortality caused by E23-2 at 10⁸ and 10¹⁰ cfu. mL⁻¹, Carbosulfan at 0.25 g/L, and E23-9 at 10¹⁰ cfu. mL⁻¹. The lowest percentage of adult mortality observed at 8 days after treatment was again achieved by the lowest rate, E25-12 at 10² cfu. mL⁻¹ (19.2% mortality). One day after the introduction of treated B. vulgaris young leaves, the greatest adult mortality was achieved by E23-2 at 10⁸ and 10¹⁰ cfu. mL⁻¹, E23-9 at 10⁸ and 10¹⁰ cfu. mL⁻¹, E23-3 at 10¹⁰ cfu. mL⁻¹, and Carbosulfan at 0.25 g/L. By 3 days after the introduction, significantly greater mortality was achieved by only E23-2 at 10⁸ and 10¹⁰ cfu. mL⁻¹, and Carbosulfan at 0.25 g/L, with mortality rates of 68.3, 76.7, and 67.5% mortality, respectively. The highest mortality percentages at 8 DAT were observed among adults exposed to B. vulgaris young leaves treated with E23-2 at 10⁸ and 10¹⁰ cfu. mL⁻¹, E23-9 at 10⁸ and 10¹⁰ cfu. mL⁻¹ and Carbosulfan at 0.25 g/L with mortality rates of 79.2, 81.7, 69.2, 77.5, and 80.8%, respectively. The tested entomopathogenic (EP) bacteria decreased insect movement before causing mortality and altered the cadavers’ color, turning them dark brown instead of bright green. Additionally, immediately after treatment, the larvae reduced in size and became smoother, while the adults’ elytra started drying out and disappearing.

The susceptibility of C. vittata to specific strains and concentrations of EP bacteria significantly influences LC50 levels.

Table 4. Median lethal concentration LC₅₀ (CFU mL⁻¹) of C. vittata treated by four different strains of Streptomyce sp. (ANOVA, α = 0.05).

Lab Bioassays	Streptomyces sp. Strains	DAT	Slope ± SE		Z		LC%50		Chi-Test (χ2) Sig (df = 58)
			Larva	Adult	Larva	Adult	Larva	Adult	Larva	Adult
Topical contact	E23-2	1	0.1 ± 0.02	0.2 ± 0.02	7.6	8.3	3.6 × 106	7.0 × 107	23.3	21.7
		3	0.2 ± 0.02	0.2 ± 0.02	10.1	10.0	8.7 × 103	1.2 × 105	29.3	25.2
		8	0.2 ± 0.02	0.2 ± 0.02	8.9	10.0	323.5	5.1 × 103	20.8	34.4
	E23-9	1	0.1 ± 0.02	0.2 ± 0.02	7.6	7.9	4.9 × 107	9.8 × 108	16.4	27.2
		3	0.2 ± 0.02	0.2 ± 0.02	8.6	8.1	1.3 × 105	3.9 × 106	15.8	21.5
		8	0.2 ± 0.02	0.2 ± 0.02	9.2	8.9	3.5 × 103	4.7 × 104	22.6	25.9
	E23-3	1	0.2 ± 0.02	0.2 ± 0.02	8.0	8.5	2.5 × 108	7.2 × 109	18.7	19.1
		3	0.2 ± 0.02	0.2 ± 0.02	8.8	7.8	6.0 × 105	3.8 × 107	27.2	21.1
		8	0.2 ± 0.02	0.2 ± 0.02	9.3	8.2	2.8 × 104	8.2 × 105	19.4	18.0
	E25-12	1	0.2 ± 0.02	0.2 ± 0.03	7.4	7.6	9.4 × 109	3.3 × 1011	22.0	36.7
		3	0.1 ± 0.02	0.2 ± 0.02	7.5	7.6	6.2 × 106	2.4 × 109	34.9	20.6
		8	0.2 ± 0.02	0.2 ± 0.02	10.5	8.5	1.7 × 105	2.7 × 107	31.3	13.6
Leaf dip	E23-2	1	0.2 ± 0.02	0.2 ± 0.02	7.7	8.5	4.3 × 107	5.6 × 108	33.8	36.8
		3	0.2 ± 0.02	0.2 ± 0.02	9.9	9.9	5.9 × 104	8.6 × 105	35.8	42.3
		8	0.2 ± 0.02	0.2 ± 0.02	8.6	9.5	1.9 × 103	3.1 × 104	25.6	39.5
	E23-9	1	0.2 ± 0.02	0.2 ± 0.02	8.0	8.3	3.8 × 108	6.3 × 109	31.5	53.0
		3	0.2 ± 0.02	0.2 ± 0.02	8.6	8.2	1.1 × 106	4.0 × 107	23.7	36.5
		8	0.2 ± 0.02	0.2 ± 0.02	9.0	8.9	2.4 × 104	3.1 × 105	34.2	35.2
	E23-3	1	0.2 ± 0.02	0.2 ± 0.03	8.3	9.0	1.9 × 109	2.1 × 1010	27.8	46.8
		3	0.2 ± 0.02	0.2 ± 0.02	9.0	8.0	4.7 × 106	3.7 × 108	37.5	33.9
		8	0.2 ± 0.02	0.2 ± 0.02	9.2	8.2	2.2 × 105	6.5 × 106	21.2	27.1
	E25-12	1	0.2 ± 0.02	0.2 ± 0.03	7.9	7.5	4.6 × 1010	9.2 × 1011	49.7	48.0
		3	0.1 ± 0.02	0.2 ± 0.02	7.6	8.0	7.4 × 107	1.4 × 1010	50.0	37.4
		8	0.2 ± 0.02	0.2 ± 0.02	10.4	8.8	1.1 × 106	1.7 × 108	48.8	21.8

DAT: Day after the treatment.

provides information on the concentrations required to achieve 50% mortality in both larvae and adults when exposed to the tested Streptomyces sp. strains. The Probit analysis, used to assess the mortality results in both methods (topical contact and the leaf dip method), revealed that the E23-2 strain was the most toxic, exhibiting the lowest median lethal concentration values of 323.5 CFU mL⁻¹ (larvae) and 5.1 × 10³ CFU mL⁻¹ (adults) for topical contact, and 1.9 × 10³ CFU mL⁻¹ (larvae) and 3.1 × 10⁴ CFU mL⁻¹ (adults) for the leaf dip method, respectively, while E25-12 had the highest values (Table 4).

In both tested methods (topical contact and the leaf dip method), one-way ANOVA analysis indicates that mean mortality was significantly (p ≤ 0.05) affected by the exposure of C. vittata larvae (

Table 5. Mortality (%), mean survival time, and LT₅₀ (days) of C. vittata larvae-treated four different strains of Streptomyces sp.

Lab Bioassays	Survival Analysis	Streptomyces sp. Strains	N c	Concentrations (CFU mL−1)
				102	104	106	108	1010
Topical contact	Mortality (%) a	E23-2	120	35.3	47.8	63.6	76.7	82.5
		E23-9	120	30.6	41.7	52.2	68.3	77.5
		E23-3	120	25.6	36.1	46.9	63.6	74.2
		E25-12	120	21.7	30.3	39.7	57.2	68.1
	Mean survival time ± SE b	E23-2	120	5.1 ± 0.1	4.8 ± 0.1	4.4 ± 0.1	4.0 ± 0.1	3.8 ± 0.1
		E23-9	120	5.2 ± 0.1	4.9 ± 0.1	4.7 ± 0.1	4.3 ± 0.1	4.0 ± 0.1
		E23-3	120	5.3 ± 0.8	5.1 ± 0.1	4.8 ± 0.1	4.4 ± 0.1	4.1 ± 0.1
		E25-12	120	5.4 ± 0.1	5.2 ± 0.1	5.0 ± 0.1	4.6 ± 1.1	4.3 ± 0.1
	LT50 (95% CI)	E23-2	120	6.0 ± 0.3	6.0 ± 0.2	6.0 ± 0.2	3.0 ± 0.2	3.0 ± 0.2
		E23-9	120	6.0 ± 0.0	6.0 ± 0.3	6.0 ± 0.2	6.0 ± 0.1	3.0 ± 0.2
		E23-3	120	6.0 ± 0.0	6.0 ± 0.3	6.0 ± 0.3	6.0 ± 0.2	3.0 ± 0.2
		E25-12	120	6.0 ± 0.0	6.0 ± 0.0	6.0 ± 0.3	6.0 ± 0.2	6.0 ± 0.1
Leaf dip	Mortality (%) a	E23-2	120	29.4	41.7	57.8	70.8	76.7
		E23-9	120	24.7	35.8	46.4	60.6	72.2
		E23-3	120	19.7	30.3	41.4	57.8	68.6
		E25-12	120	15.2	24.4	33.9	51.4	62.2
	Mean survival time ± SE b	E23-2	120	5.3 ± 0.1	4.9 ± 0.1	4.5 ± 0.1	4.2 ± 0.1	4.0 ± 0.1
		E23-9	120	5.4 ± 0.1	5.1 ± 0.1	4.8 ± 0.1	4.5 ± 0.1	4.1 ± 0.1
		E23-3	120	5.5 ± 0.1	5.3 ± 0.1	4.9 ± 0.1	4.5 ± 0.1	4.2 ± 0.1
		E25-12	120	5.6 ± 0.7	5.4 ± 0.1	5.1 ± 0.1	4.7 ± 0.1	4.5 ± 0.1
	LT50 (95% CI)	E23-2	120	6.0 ± 0.0	6.0 ± 0.3	6.0 ± 0.2	6.0 ± 0.1	3.0 ± 0.2
		E23-9	120	6.0 ± 0.0	6.0 ± 0.3	6.0 ± 0.2	6.0 ± 0.2	6.0 ± 0.1
		E23-3	120	6.0 ± 0.0	6.0 ± 0.0	6.0 ± 0.3	6.0 ± 0.2	6.0 ± 0.1
		E25-12	120	6.0 ± 0.0	6.0 ± 0.0	6.0 ± 0.3	6.0 ± 0.2	6.0 ± 0.1

^a Abbott-corrected percentage mortality of C. vittata larvae at the end of experiment; ^b the mean survival time and its standard error; ^c total number of scale insects in bioassay.

) and adults (

Table 6. Average numbers of Cassida vittata larvae per 20 Beta vulgaris plants at 1 day before treatment and at 3, 6, and 12 days after treatments (DAT) with Streptomyces sp. strains, insecticide, or tap water under field conditions.

Treatments	Mean Density (± SEM) at				p
	1 d-Pre-trt	3 DAT	6 DAT	12 DAT
E23-2 at 108 CFU mL−1	210.8 ± 0.6 ABa	52.3 ± 2.1 Cb	39.3 ± 2.3 Cc	29.3 ± 2.3 Dd	p < 0.0001
E23-2 at 1010 CFU mL−1	210.3 ± 0.8 ABa	45.2 ± 2.5 Db	32.2 ± 0.8 Dc	22.2 ± 0.8 Ed	p < 0.0001
E23-9 at 108 CFU mL−1	214.0 ± 3.9 Aa	62.3 ± 2.9 Bb	48.3 ± 2.9 Bc	45.5 ± 2.3 Bc	p < 0.0001
E23-9 at 1010 CFU mL−1	210.7 ± 2.2 ABa	55.2 ± 2.4 Cb	41.5 ± 1.1 Cc	39.8 ± 1.0 Cc	p < 0.0001
Carbosulfan at 0.25 g/L	210.3 ± 3.3 ABa	46.0 ± 2.2 Db	33.3 ± 1.8 Dc	31.2 ± 1.3 Dc	p < 0.0001
Control	210.8 ± 1.7 ABc	215.7 ± 1.4 Ab	219.0 ± 2.2 Ab	224.7 ± 2.9 Aa	p < 0.0001
Statistical analysis	F = 9.6, df = 5, 30 p < 0.0001	F = 5195.8, df = 5, 30 p < 0.0001	F = 8562.0, df = 5, 30 p < 0.0001	F = 10,189.6, df = 5, 30 p < 0.0001

Within columns means followed by the same capital letters are not statistically different according to Tukey’s LSD test at α = 0.05. Within lines means followed by the same lower-case letters are not statistically different according to Tukey’s LSD test at α = 0.05.

) to varying concentrations of EP-bacteria suspensions. Insects exposed during the period from 1 to 8 days after treatment to the highest concentration (10¹⁰ cfu. mL⁻¹) exhibited a significantly higher mortality rate. The mean survival time of C. vittata larvae exposed to select EP-bacteria with different concentrations ranged from a minimum of 3.8 days in E23-2 at 10¹⁰ cfu. mL⁻¹ to a maximum of 5.4 days in E225-12 at 10² cfu. mL⁻¹ in the topical contact method and from a minimum of 4.0 days in E23-2 at 10¹⁰ cfu. mL⁻¹ to a maximum of 5.6 days in E225-12 at 10² cfu. mL⁻¹ in the leaf dip method (Table 5). In the topical contact method, the mean survival time of C. vittata adults ranged from a minimum of 4.0 days in E23-2 at 10¹⁰ cfu. mL⁻¹ to a maximum of 5.8 days in E225-12 at 10² cfu. mL⁻¹. In the leaf dip method, the mean survival time values of C. vittata adults ranged from a minimum of 4.1 days in E23-2 at 10¹⁰ cfu. mL⁻¹ to a maximum of 5.9 days in E225-12 at 10² cfu. mL⁻¹ (Table 6). In terms of median survival time (LT₅₀), the lowest value of LT₅₀ (3 days) was observed in E23-2, E23-3, and E23-9 at 10¹⁰ cfu. mL⁻¹, and E23-2 at 10⁸ cfu. mL⁻¹ in the topical contact method. Additionally, only E23-2 at 10¹⁰ cfu. mL⁻¹ in the leaf dip method exhibited a similar LT₅₀ value of 3 days. The highest LT₅₀ value (6 days) was recorded in the other treatments tested for larvae (Table 5). For adults, the lowest LT₅₀ value (3 days) was observed in E23-2 at 10¹⁰ cfu. mL⁻¹ in the topical contact method, with no significant difference recorded among the other treatments (6 days). No difference was recorded among the tested treatments (6 days) in the leaf dip method for adults (Table 5).

3.3. Repellent Activity

All four Streptomyces sp. strains assessed repelled C. vittata adults. The repulsion index (RI) of the four strains tested at 10¹⁰ cfu. mL⁻¹ was presented in Figure 1. The results showed that all strains tested had a higher repellent effect 48 h after application. The E23-2 strain provided the highest repellent effect at 12 h (50.6%), 24 h (63.4%), and 48 h (84.9%) after application, followed by E23-9 with repellence index values of 12 h (42.4%), 24 h (61.6%), and 48 h (81.3%), and E23-3 with repellence index values of 12 h (25.7%), 24 h (42.7%), and 48 h (60.9%), while E25-12 had a low repellent effect on C. vittata adults with RI values of 12 h (5.6%), 24 h (8.9%), and 48 h (22.2%).

3.4. Field Trials

The efficacy of Streptomyces sp. strains in controlling C. vittata larvae on B. vulgaris plants under field conditions was investigated (Table 6). Both E23-2 and E23-9 strains displayed significant effectiveness in reducing larvae density. Specifically, E23-2 at 10¹⁰ cfu. mL⁻¹ exhibited a substantial decrease from 210.8 larvae 1 day before treatment to 22.2 at 12 days after treatment. E23-9 also showed a notable reduction, with higher concentrations yielding more pronounced effects. A concentration-dependent effect was evident for both strains, highlighting the importance of dosage for optimal control. Carbosulfan, the chemical insecticide, demonstrated efficacy comparable to or slightly less than the higher concentration of Streptomyces sp. strains.

Table 7. Average numbers of Cassida vittata adults per 20 Beta vulgaris plants at 1 day before treatment and at 3, 6, and 12 days after treatments (DAT) with Streptomyces sp. strains, insecticide, or tap water under field conditions.

Treatments	Mean Density (± SEM) at				p
	1 d-Pre-trt	3 DAT	6 DAT	12 DAT
E23-2 at 108 CFU mL−1	181.5 ± 34.7 Aa	55.7 ± 1.2 Cb	42.3 ± 2.3 Cb	30.0 ± 1.7 Eb	p < 0.0001
E23-2 at 1010 CFU mL−1	185.8 ± 37.6 Aa	47.7 ± 2.7 Db	35.5 ± 1.4 Db	23.7 ± 1.2 Fb	p < 0.0001
E23-9 at 108 CFU mL−1	210.5 ± 3.7 Aa	63.3 ± 2.3 Bb	50.2 ± 2.9 Bc	47.0 ± 1.7 Bc	p < 0.0001
E23-9 at 1010 CFU mL−1	207.5 ± 3.7 Aa	56.3 ± 2.3 Cb	42.8 ± 1.2 Cc	40.7 ± 1.2 Cc	p < 0.0001
Carbosulfan at 0.25 g/L	203.2 ± 3.8 Aa	48.3 ± 2.5 Db	35.8 ± 1.2 Dc	33.7 ± 1.2 Dc	p < 0.0001
Control	209.7 ± 1.9 Ac	214.7 ± 1.9 Ab	217.0 ± 1.8 Ab	223.3 ± 2.3 Aa	p < 0.0001
Statistical analysis	F = 2.2, df = 5, 30 p < 0.0001	F = 4923.2, df = 5, 30 p < 0.0001	F = 8673.9, df = 5, 30 p < 0.0001	F = 14,283.4, df = 5, 30 p < 0.0001

Within columns means followed by the same capital letters are not statistically different according to Tukey’s LSD test at α = 0.05. Within lines means followed by the same lower-case letters are not statistically different according to Tukey’s LSD test at α = 0.05.

illustrates the impact of treatments on C. vittata adult populations in B. vulgaris plants. Both concentrations of Streptomyces sp. strains show a decreasing trend in adult densities over time, with higher concentrations yielding lower densities. Three days after treatment, E23-2 at 10¹⁰ cfu. mL⁻¹ significantly reduces density to 47.7 adults per 20 plants compared with the control group with 214.7 adults. Carbosulfan at 0.25 g/L also proves effective, with a density of 48.3 adults at 3 days after treatment. The control group experienced a notable increase to 217.0 adults at 8 days after treatment. Statistical analyses confirm significant differences between treatments at each time point, highlighting the efficacy of Streptomyces sp. strains and Carbosulfan in reducing C. vittata adult populations compared with the control group.

The Henderson–Tilton adjusted rates of population reduction for C. vittata larvae and adults at 12 days post-treatment (

Table 8. Henderson–Tilton adjusted rates of population reduction in Cassida vittata at 12 days after treatments with Streptomyces sp. strains, insecticide, or tap water under field conditions.

Treatments	Mean % Reduction (± SEM) of
	Larva	Adult
E23-2 at 108 CFU mL−1	84.8 ± 1.2 Ba	81.9 ± 3.6 Ba
E23-2 at 1010 CFU mL−1	88.6 ± 0.4 Aa	85.9 ± 3.0 Aa
E23-9 at 108 CFU mL−1	77.4 ± 0.9 Da	76.2 ± 0.6 Cb
E23-9 at 1010 CFU mL−1	79.9 ± 0.6 Ca	79.0 ± 0.7 BCa
Carbosulfan at 0.25 g/L	84.1 ± 0.9 Ba	82.3 ± 0.8 Bb
Statistical analysis	F= 169.3, df = 4, 25, p < 0.0001	F = 18.9, df = 4, 25, p < 0.0001

Within columns means followed by the same capital letters are not statistically different according to Tukey’s LSD test at α = 0.05. Within lines means followed by the same lower-case letters are not statistically different according to Student’s t-test, p < 0.05.

) reveal promising results. Streptomyces sp. strains, notably E23-2 at 10¹⁰ cfu. mL⁻¹, exhibit substantial reductions in larval (88.6%) and adult (85.9%) populations. Carbosulfan at 0.25 g/L also demonstrates effectiveness, yielding reductions of 84.1% (larvae) and 82.3% (adults). Table 8 also highlights distinct responses to different tested treatments between C. vittata larvae and adults. In the E23-9 at 10⁸ cfu. mL⁻¹ treatment, larval reduction (77.4%) significantly surpasses adult reduction (76.2%) (t = 2.570, df = 10, p = 0.028). Likewise, within the Carbosulfan at 0.25 g/L treatment, larval reduction (84.1%) exceeds adult reduction (82.3%) (t = 3.644, df = 10, p = 0.005). No significant difference between larvae and adults is recorded for other treatments. These findings emphasize the differential impact of treatments on distinct life stages of C. vittata.

The results in

Table 9. Functional significance of the Streptomyces sp. strains selected.

Streptomyces sp. Strains	Chitinase Production	Cellulase Production	Protease Production
E23-2	+	+	+
E23-3	+	+	+
E23-9	+	+	+
E25-12	+	-	+

(-) Strains with no enzymatic activity, (+) strains with enzymatic activity.

reveal the enzymatic capabilities of selected Streptomyces sp. strains. E23-2, E23-3, and E23-9 exhibit positive chitinase, cellulase, and protease production, indicating their versatility for various applications. However, E25-12 lacks cellulase production, suggesting limitations in certain processes. These findings provide valuable insights for leveraging the functional attributes of Streptomyces sp. strains in biocontrol applications.

4. Discussion

Conventional insecticides were the primary method for controlling C. vittata, but their use posed issues such as toxic environmental residues, pesticide resistance, and harm to natural enemies [54]. Integrated Pest Management (IPM) has gained significance in recent times as a more holistic approach. Various microorganisms, including bacteria, have been employed as entomopathogens (EPs) for C. vittata control in numerous IPM programs worldwide [10]. The present study investigated the evaluation of four Streptomyces sp. strains as biological control agents against C. vittata, a damaging scale pest of sugar beet, and B. vulgaris L. cultivation worldwide. In the present study, all tested Streptomyces sp. strains exhibited effective growth on diverse carbon sources, with E23-3 and E25-12 demonstrating superior pH tolerance compared with E23-2 and E23-9. Additionally, the Streptomyces sp. strains showcased optimal growth within a pH range of 5.33 to 10.03, with the highest growth occurring at pH 8.28. The strains displayed a preference for neutral or slightly alkaline pH conditions, in line with their inability to grow in acidic media (pH < 5) [38]. These strains displayed robust tolerance to NaCl up to 3%, facing challenges at higher concentrations, especially at 10% NaCl, suggesting their ability to resist NaCl concentrations up to 70 g/L (7%) [55]. Moreover, the Streptomyces sp. strains tested were mesophilic and thrived at temperatures of 28 °C and 37 °C, consistent with the most favorable temperature range for their growth [56]. Similar to findings by Singh et al. [57], none of the tested Streptomyces sp. strains could grow at 4 °C and 46 °C.

In general, the interaction between microorganisms and insects spans from symbiosis and mutualism to parasitism, and certain microorganisms associated with insects have the capability to generate toxins that modify tissues, leading to the demise of the affected pests [17,58]. In this study, laboratory trials demonstrated concentration-dependent mortality trends for C. vittata larvae and adults exposed to different Streptomyces sp. strains. E23-2 at 10¹⁰ cfu. mL⁻¹ in the topical contact method induced the highest 1-day larval mortality (90.0%), comparable to Carbosulfan at 0.25 g/L. The leaf dip method showed similar trends, with E23-2 consistently causing the greatest larval mortality. For adults, both E23-2 at 10¹⁰ cfu. mL⁻¹ and Carbosulfan at 0.25 g/L resulted in the highest mortality at 8 DAT (88.3%). Rahoo et al. [59] emphasized the role of EP-bacteria concentration, highlighting its impact on infection chances and pest mortality rates. Probit analysis identified E23-2 as the most toxic, with the lowest LC₅₀ values for topical contact (323.5 CFU mL⁻¹ for larvae and 5.1×10³ CFU mL⁻¹ for adults) and the leaf dip method (1.9×10³ for larvae and 3.1×10⁴ CFU mL⁻¹ for adults). These values underscore E23-2’s potent insecticidal activity against C. vittata. Entomopathogenic bacteria play a key role in the natural mortality of insect pests [12]. The main advantages of these biocontrol agents are their specificity to target pests, safety to non-target organisms, lack of harmful effects on the environment and human health, and their effectiveness against pests that have developed resistance to conventional insecticides [60]. These qualities make them ideal components of integrated pest management (IPM) strategies and organic farming systems. The biocide Bacillus thuringiensis var. Kurstaki (Dipel 2X) reduced C. vittata populations by 19.01% in 1998/1999 and 27.78% in 1999/2000 in Egypt [10]. Other studies using entomopathogenic fungi and insecticides, both alone and in combination, showed different results. Biorational insecticides and entomopathogenic fungi were tested against C. vittata; Alternaria destruens (PP264311) (Pleosporales: Pleosporaceae) LC₅₀ was 4.2 × 10⁷ conidia. mL⁻¹ alone, decreasing to 2.2 × 10⁷ with spinosad, 1.5 × 10⁷ with mineral oil, and 3.1 × 10⁷ conidia. mL⁻¹ with potassium salts; Alternaria murispora (PP264308) (Pleosporales: Pleosporaceae) LC₅₀ was 1.3 × 10⁷ conidia. mL⁻¹ alone, increasing to 2.3 × 10⁷ with spinosad, 1.7 × 10⁷ with mineral oil, and 1.7 × 10⁷ conidia. mL⁻¹ with potassium salts [61]. Metarhizium anisopliae (Metschn.) (Hypocreales: Clavicipitaceae), Beauveria bassiana (Balsamo) (Hypocreales: Cordycipitaceae), and Verticillium lecanii (Zimm.) (Hypocreales: Clavicipitaceae) caused 47–95% egg mortality in C. vittata within seven days, with M. anisopliae causing the highest adult mortality (69%), followed by B. bassiana (65%) and V. lecanii (55%) [62]. The primary method for controlling C. vittata involved conventional insecticides such as imidacloprid, thiamethoxam, chlorpyrifos, and combinations like thiamethoxam + abamectin [54]. However, this approach led to issues such as the accumulation of toxic environmental residues, the development of pesticide-resistant pest strains, and harm to beneficial natural enemies [54,62]. The differences in efficacy among the various methods of control for C. vittata can be attributed to several factors, including the type of biocontrol agent, the method of application, and the interaction between the pest, pathogen, and environmental conditions, highlighting the importance of understanding these variables for the effective use of EP-based biopesticides in pest control [63]. Notably, adults, with their elytra-covered bodies, showed lower mortality compared with larvae, attributed to protection from direct contact. The experimental results highlight significant impacts on larval and adult mortality when exposed to varying EP-bacteria concentrations in both application methods. Streptomyces sp. are characterized by their ability to produce toxic proteins that can be harmful to insects [64]. Larvae exhibited a mean survival time ranging from 3.8 to 5.4 days in the topical contact method and from 4.0 to 5.8 days for adults. The median survival time (LT₅₀) was lowest at 3 days for specific treatments, emphasizing concentration-dependent efficacy. In the leaf dip method, similar trends revealed higher mortality rates at higher bacterial concentrations, providing valuable insights for pest management strategies. Visual changes in treated insects further indicated the impact of Streptomyces sp. strains.

Streptomyces sp. was reported to have biocontrol potential for many pest species attacking several economically important crops, such as S. littoralis [65] and P. xylostella [21]. The current investigation provides additional evidence that Streptomyces sp. causes increased mortality of the major sugar beet pest, C. vittata, and has the potential as an EP agent for the control of this harmful insect pest. The bacteria’s toxins may have affected the insect’s gut membrane, causing reduced nutrient absorption and contributing to the mortality of C. vittata [58]. All the tested Streptomyces sp. strains were found positive for chitinase and protease production. Bacterial enzymes play a crucial role in virulence, degrading the insect body for survival within the host and neutralizing its defense mechanisms [66]. Moreover, the chitinase enzyme from both fungi and bacteria has been employed to suppress larvae of bollworm (H. armigera) and various stem borers (Eldana saccharina (Wlk.), Sesamia calamistis (Hampson), and Chilo partellus (Swinhoe) (Lepidoptera–Pyralidae), disrupting their peritrophic membrane containing chitin [19,20]. In field trials, Streptomyces sp. strains, especially E23-2 at 10¹⁰ cfu. mL⁻¹, significantly reduced C. vittata larvae from 210.8 to 22.2 at 12 days post-treatment. E23-9 also showed notable reductions. Carbosulfan, a chemical insecticide, had efficacy comparable to the higher Streptomyces sp. concentration. For adults, E23-2 at 10¹⁰ cfu. mL⁻¹ significantly reduced density to 47.7 at 3 days post-treatment. Henderson–Tilton rates at 12 days showed E23-2 achieved 88.6% larval and 85.9% adult reductions. Carbosulfan demonstrated reductions of 84.1% (larvae) and 82.3% (adults). Results highlight treatment specificity for C. vittata life stages. The tested Streptomyces sp. strain employs diverse mechanisms for insect pest control, with chitin degradation considered a key factor. Chitinolytic organisms, such as Streptomyces sp. from the rhizosphere, have demonstrated potential as biological control agents under field conditions [10,38]. Other EPs-bacteria, like Pseudomonas entomophila, as reported by Vodovar et al. [67] induce mortality in Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) through robust hemolytic activity involving proteins like lipases and chitinases. Bacterial chitinases, known to hydrolyze chitinous exoskeletons, have proven effective against insects and mites [68]. Wilson et al. [69] highlighted the role of bacterial hemolysins in attacking blood cell membranes, possibly contributing to the rapid mortality observed in C. vittata with E23-2 at 10¹⁰ cfu. mL⁻¹. Vodovar et al. [67] also suggested that pathogenic bacteria rely on cell surface-associated virulence factors for effective colonization, potentially enhancing the infiltration of proteases, chitinases, lipases, and hydrolases, leading to rapid mortality in C. vittata. All four Streptomyces sp. strains effectively repelled C. vittata adults at 10¹⁰ cfu. mL⁻¹, with E23-2 showing the highest repellent effect (84.9% at 48 h). The observed repellent effect of Streptomyces sp. strains is attributed to the production of secondary metabolites, particularly volatile compounds, which effectively deter insects from treated leaves [70].

Research on the control of C. vittata with entomopathogenic (EP) bacteria is limited, and EP bacteria-derived biomolecules show promising compatibility under various conditions [38]. Integration into IPM programs requires precise identification and understanding of the bioecology, behavior, and effects on non-target insects [71]. Molecular tools for distinguishing strains and detecting EPs are crucial. Streptomyces sp. species are known for producing bioactive secondary metabolites, some of which exhibit insecticidal properties [72]. These metabolites, such as blasticidin-S, kusagamycin, streptomycin, oxytetracycline, validamycin, polyoxins, natamycin, actinovate, mycostop, abamectin/avermectins, emamectin benzoate, polynactins, and milbemycin, have been widely applied in agriculture as biocontrol agents due to their effectiveness in pest management [73]. Their specificity makes them particularly attractive, as they are generally safer for non-target organisms, including beneficial insects, mammals, and humans [74]. These Streptomyces-derived compounds present a promising alternative for managing insect pests and plant pathogens, reducing environmental and non-target impacts [73]. In the present study, we have not directly assessed the safety of Streptomyces strains to B. vulgaris. However, secondary metabolites from Streptomyces are recognized as a key group of new-generation pesticides in integrated pest management (IPM) due to their targeted toxicity and minimal risk to non-target organisms and ecosystems [75]. Furthermore, their use has gained public approval due to their low environmental footprint and safety for humans and other non-target species [29]. Streptomyces sp. exhibits high toxicity to mosquitoes while being less harmful to non-target organisms [64]. Commercially used Streptomyces-derived secondary metabolites, known for their specificity and safety, offer an eco-friendly alternative for pest and pathogen management [73]. Streptomyces secondary metabolites present a promising new-generation pesticide with minimal impact on ecosystems and humans [29]. The synergistic effects of secondary metabolites may contribute to the high insecticidal activity observed.

5. Conclusions

In conclusion, the Streptomyces sp. strains tested, particularly E23-2, exhibit promising potential as biocontrol agents against C. vittata. Their concentration-dependent efficacy, minimal impact on non-target organisms, and suitability for IPM highlight their significance. However, environmental variability, including fluctuations in temperature, humidity, and soil composition, may influence their field performance. Additionally, long-term studies are needed to assess their persistence and interactions with native microbial communities. Future research should explore molecular tools for strain identification, optimize application strategies, and investigate potential synergies between Streptomyces sp. metabolites and other IPM components. This study contributes to sustainable pest management and aligns with the global push for eco-friendly agricultural practices. The integration of Streptomyces sp. into IPM programs presents a valuable avenue for effective and environmentally conscious pest control.