Synergistic effect of naturally occurring Granulosis virus isolates (PbGV) with phagostimulants against the cabbage butterfly, Pieris brassicae (L.) for its eco-friendly management

Background To manage the cabbage butterfly, Pierisbrassicae (L.) (Lepidoptera: Pieridae), it is not wise to use insecticides on leafy vegetables which are eaten mostly fresh. During the past decades, the efforts to manage the pest, through chemical insecticides have raised serious health. Investigations were carried out to isolate naturally occurring GVs (PbGV) as a potent biopesticide against P.brassicae and to explore their efficacy with the application of phagostimulants. Results Among the four naturally occurring isolates obtained from Northwestern Himalayas, Sudhmahadev isolate was found to be the most promising based on virulence and speed of kill against all the instars tested in the laboratory, showing the natural incidence of PbGV infection in field conditions. In concentration and time–response bioassay, all the isolates of P.brassicae Granulosis virus were found high virulent against second instar larvae of cabbage butterfly. Therefore, for enhanced efficacy of PBGV, its combined application with phagostimulants (Lepidiumsativum + Teepol + jaggery) or sticker (Teepol + jaggery), applied in field trials, resulted into greater mortality of larval instars than the single one. Overall, the results indicated that the introduction of a more isolates PBGV strain into populations of P.brassicae could be of vital importance for eco-friendly suppression of this pest globally with the combination of phagostimulants. The application virus alone with the pre-standardized concentration of 1 × 1012 OBs/ha did not reduce the larval population density to the desirable extent in the greenhouse chamber and therefore was not included in field experiments. Overall, the most promising treatments in reducing the larval population of the pest were PbGV + Teepol + B.thuringiensis (93.49 and 91.39%) and PbGV + Teepol + L.sativum (88.79 and 86.97%) over control in both greenhouse and field trials, respectively. Conclusions In this study, the native isolates of PbGV from different target locations to test their efficacy against different instars of P.brassicae were explored. Using native PBGV isolates with phagostimulant combinations played an important role for regulating the pest effectively. These phagostimulants not only protected the OBs from degradation in the presence of sunlight but also increased the speed of killing. The biocontrol potential of PbGV in both laboratory and field conditions indicated that baculoviruses are sustainable alternative to chemical insecticides.

Efficacy of Beauveria bassiana and Beauveria pseudobassiana isolates against the pine processionary moth, Thaumetopoea wilkinsoni Tams, 1926 (Lepidoptera/Notodontidae)

Background The pine processionary moth, Thaumetopoea wilkinsoni Tams, 1926 (Lepidoptera/Notodontidae) is one of the most harmful insects that destroys pine ecosystems by feeding on pine leaves at its larval stage. Because of its urticating setae, the insect also causes severe skin reactions to animals and humans. Instead of chemical control, eco-friendly biological control methods are preferred to combat this species. Results The purpose of this study was to evaluate the efficacy of five different Beauveria bassiana Vuill, 1912 (Hypocreales/Cordycipitaceae) isolates (TR-SM-10, TR-SM-11, TR-SM-2, TR-SK-1 and TR-D-1) and one B. pseudobassiana Rehner & Humber (Hypocreales/Cordycipitaceae) isolate (TR-SM-1) against the fourth instar larvae of T. wilkinsoni under laboratory conditions. T. wilkinsoni larvae were collected from the Ondokuz Mayıs University Kurupelit Campus in Samsun, Turkey, in 2021, and the fourth instar larvae were used in the experiment. Two ml of spray of the six fungal isolates were applied to every ten larvae at each concentration (1 × 107 and 1 × 108 conidia ml−1). The experiment was carried out in five replicates per group, and the larvae were observed for 10 days. As a result, all isolates of B. bassiana caused 100% mortality at 1 × 108 conidia ml−1 concentration. B. pseudobassiana isolate also caused 100% mortality at both concentrations. At 1 × 107 conidia ml−1 concentration, the larvae treated with the B. pseudobassiana isolate (TR-SM-1) had the lowest LT50 (2.89 days) and LT90 values (4.79 days), while the larvae treated with TR-SM-10 isolate had the highest LT50 (5.65 days) and LT90 values (9.39 days). At 1 × 108 conidia ml−1 concentration, the larvae treated with TR-SK-1 isolate had the lowest LT50 (2.89 days) and LT90 values (4.79 days), while those treated with TR-SM-10 isolate had the highest LT50 (3.95 days) and LT90 values (8.15 days). Conclusion It has been recommended that the five different isolates of B. bassiana and B. pseudobassiana isolates were virulent to T. wilkinsoni larvae and can be used for biological control of T. wilkinsoni.

Effect of endophytic Bacillus and arbuscular mycorrhiza fungi (AMF) against Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici

Background Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici (FOL) is a serious disease that causes significant economic losses in tomato production. Seventeen endophytic Bacillus isolates from tomato roots of Meghalaya were tested for antagonistic and plant growth promotion activities. Dominating arbuscular mycorrhiza fungi (AMF) spores were isolated from the rhizosphere soils of tomato grown in Meghalaya. The effect of different combinations of AMF and endophytic Bacillus on Fusarium wilt severity and growth of tomato plant under pot and field conditions was studied. Results The endophytic Bacillus isolates ERBS51 and ERBS10 showed a maximum inhibition against FOL, with 58.43 and 55.68%, respectively, in a dual culture experiment. ERBS51 and ERBS10 were identified as Bacillus velezensis and Bacillus sp., respectively, based on 16s rRNA sequencing. Both isolates were found positive for iturin A, surfactin, bacillomycin D, protease, cellulase, pectinase, alpha-amylase, siderophore, ammonia production and ZnCO3 solubilization. Funneliformis mosseae and Glomus fasciculatum were the dominating AMF species in tomato rhizosphere of Meghalaya. The result of pot and field experiments revealed that out of all the treatments, combination of Funneliformis mosseae + Glomus fasciculatum + Bacillus velezensis + Bacillus sp. was shown to be the best in reducing the severity of Fusarium wilt to 77.44 and 66.74%, respectively. F. mosseae + G. fasciculatum + B. velezensis + Bacillus sp. also recorded the highest in most growth attributes and yield. Conclusions Endophytic Bacillus (B. velezensis and Bacillus sp.) and AMF (F. mosseae and G. fasciculatum) were safe and effective biocontrol agents against Fusarium wilt of tomato.

Efficacy of the entomopathogenic nematode isolate Heterorhabditis taysearae to control the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

Background Entomopathogenic nematodes (EPNs) are promising alternatives since they have many characteristics as a biological control agent against insect pests. Results Among indigenous EPN isolated, adapted to local environmental conditions by employing the Galleria baiting strategy, only one sample was positive for the presence of EPNs. The new isolate was identified at the species level using DNA sequencing of the internal transcribed spacer region and the Basic Local Alignment Search Tool search of GenBank showed that the isolate had a high similarity (99%) with that sequence available for Heterorhabditis taysearae. The pathogenicity of the EPN isolate was tested against the cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), using different concentrations (60, 90, 120 and 150 IJs/larvae). Data showed that 150 IJs/larvae caused 100% mortality rate, followed by 120 IJs/larvae (90%), while 60 IJs/larvae showed the lowest rate (60%) after 72 h. Conclusions The present study indicated that the native isolate of EPN could be recommended against S. littoralis as an efficient tool in its control programs.

Isolation, identification and virulence of indigenous entomopathogenic fungal strains against the peach-potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), and the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

Background As different biogeographic strains and isolates of entomopathogenic fungi vary in their genetic, enzymatic and pathogenic characteristics, this study assessed the virulence of 2 indigenous strains of Beauveria bassiana (Balsam) Vuillemin and Metarhizium anisopliae (Metschn.) Sorokin (Ascomycota, Hypocreales: Clavicipitaceae), isolated from naturally infected insect cadavers, against the 3rd instar nymphs of Myzus persicae (Sulzer) (Hemiptera: Aphididae) and 3rd instar larvae of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) using leaf-dip and larval-dip methods, respectively. Results Both fungal isolates exhibited considerable pathogenicity against M. persicae and S. frugiperda. Mortality in all bioassays was conidial concentration and exposure time dependent and increased significantly along with both factors (R2 = 0.86–0.99 for B. bassiana and 0.82–0.94 for M. anisopliae). Moreover, M. anisopliae isolate appeared more virulent to S. frugiperda larvae than B. bassiana isolate, while the later fungal isolate was more lethal to M. persicae nymphs than the former one. At the highest conidial concentration (1.0 × 109 conidia/ml), M. anisopliae caused maximum mean mortality of S. frugiperda (88%) and M. persicae (65%) and B. bassiana exhibited maximum mean mortality of S. frugiperda (76%) and M. persicae (94%). Moreover, probit regression analyses showed LT50 values for M. persicae of 4.57 and 6.86 days at 1.0 × 109 conidia/ml for the isolates of B. bassiana and M. anisopliae, respectively, while LC50 values were 7.75 × 106 and 8.70 × 107 conidia/ml after 10th day of application, for the isolates of B. bassiana and M. anisopliae, respectively, against M. persicae. Similarly, LT50 values for S. frugiperda were 7.75 and 7.03 days for 1.0 × 109 conidia/ml concentration and LC50 values were 2.84 × 107 and 8.84 × 105 conidia/ml at 10th day data for the isolates of B. bassiana and M. anisopliae, respectively. Conclusion Overall study results demonstrated the effectiveness of B. bassiana and M. anisopliae against M. persicae and S. frugiperda, respectively. However, field evaluations of these indigenously isolated promising fungal strains against these insect pests.

Compatibility of entomopathogenic nematodes with insecticides against the cabbage white butterfly, Pieris rapae L. (Lepidoptera: Pieridae)

Background Compatibility of entomopathogenic nematodes (EPNs) with insecticides is a crucial mainstay of integrated pest management (IPM) programs. This study was designed to evaluate the joint action of EPN species and insecticides when employed to deter 3rd and 4th larval instars of cabbage white butterfly, Pieris rapae L. (Lepidoptera: Pieridae) under laboratory conditions. EPNs [Steinernema carpocapsae (All strain), S. feltiae (Filipjev), Heterorhabditis bacteriophora (HP88), and H. bacteriophora (Ar-4)], at concentrations of 50, 100, and 125 IJs/larva, were tested with 3 insecticides (lambda-cyhalothrin, emamectin benzoate, and indoxacarb) at LC25 and LC50 values. Additionally, expression profiles of 2 detoxification genes (CYP6AE120 and PrGSTs1) when the 4th instar larvae were treated by H. bacteriophora (HP88) and lambda-cyhalothrin were examined. Results Data indicated that statistically significant mortality of 2 larval instars of P. rapae was observed in vitro among EPN species and pesticide concentrations. At concentration of 50 IJs/larva, LT50 values were 2.385 and 3.92 days for S. carpocapsae (All strain) and H. bacteriophora(Ar-4), respectively, on 3rd instar larvae; also, these values were 3.506 and 3.107 days for S. feltiae and H. bacteriophora (Ar-4), respectively, on 4th instar larvae Lambda-cyhalothrin was the most toxic insecticide, followed by emamectin benzoate and indoxacarb at LC25 and LC50, respectively. An additive effect was observed between EPN species with LC25 and LC50 of the tested insecticides, except for lambda-cyhalothrin at LC50 with H. bacteriophora (Ar-4), and indoxacarb, with all EPNs showing antagonistic effects on mortality of 3rd instar larvae after 3 days post-treatment. The interaction between the tested pesticides at LC25 and LC50 and EPN species, showed an additive effect, excluding lambda-cyhalothrin at LC25 with S. carpocapsae (All strain) and LC25 of indoxacarb with H. bacteriophora (Ar-4), which showed potentiation effects. The interaction of S. feltiae (Filipjev) with tested insecticides at LC50 exhibited an antagonistic effect on the mortality of 4th instar P. rapae larvae after 3 days post-treatment. The expression of both CYP6AE120 and PrGSTs1 was significantly up-regulated with lambda-cyhalothrin, followed by H. bacteriophora (HP88) compared to control. Conclusions The findings suggested that combining EPNs and the pesticide concentrations can be a practical strategy for managing P. rapae and could pave the way to using new control technologies in protecting organic farm vegetables from lepidopteran pests.

Characterization of endophytic yeast and its suppressive effect on root rotting fungi of tomato under neem cake soil amendment

Background The exposure of crops to a variety of fungal and bacterial pathogens leads to huge economic losses. Different strategies are being adapted to control these diseases among which the application of chemicals fungicide is common. However, these chemicals are posing a serious threat to the environment. For biological management of root rot disease of tomato and better fruit quality, studies were conducted on the possible use of endophytic yeast as a biocontrol agent. Results Endophytic yeasts were isolated from healthy plants and identified. Identification of selected isolates was confirmed on the basis of 18S rDNA gene sequencing. They were evaluated for suppressive effect on root rotting fungi in vitro and also in vivo on tomato plants, used alone or under neem cake soil amendment. Seventy-six isolates of yeasts were evaluated against root rotting fungi Fusarium oxysporum, F. solani, Rhizoctonia solani and Macrophomina phaseolina using dual culture plate assay. Seventy-five isolates were found to suppress radial growth of F. oxysporum, F. solani and M. phaseolina by producing zones of inhibition or lysing the fungal hyphae. However, none of the isolates was found to inhibit R. solani in vitro. Most of the isolates also caused nematicidal activity at varying degree against Meloidogyne javanica. All test isolates produced indole acetic acid in vitro and solubilized phosphorus. In pots and field plot experiments, test isolates of yeasts were able to suppress root rotting fungi on tomato in natural soil and soil amended with neem cake with enhancement of growth of tomato plants. Yeasts were also found to ameliorate the plant resistance through enhancing polyphenolic contents, salicylic acid and antioxidant activity. Conclusions Endophytic yeasts were found effective against root rot disease of tomato and could be used as a potential biocontrol agent for the management of soil-borne diseases of tomatoes.

Diversity of indigenous Bacillus thuringiensis isolates toxic to the diamondback moth, Plutella xylostella (L.) (Plutellidae: Lepidoptera)

Background Toxins from the Bacillus thuringiensis (Bt) bacterium are employed as an alternative to synthetic pesticides in pest management. The greatest threat to the long-term viability of Bt toxins is resistance evolution in the target pests. Genetic diversity and toxicity of Bt isolates were studied in this work in order to find Bt isolates with novel cry genes. Results In terms of colony morphology, among a total of 60 isolates, 51 isolates had off-white colour colonies with typical fried egg appearance, irregular shape, flat and undulate margin. Different crystal shapes, viz. spherical (88.13%), bipyramidal (49.15%), cuboidal (42.37%), rectangular, and crystals attached to spores (3.38%) were observed among Bt isolates. SDS-PAGE analysis of spore crystal mixture showed the presence of proteins with various molecular weights ranging from 124 to 26 kDa. PCR screening with cry1, cry2, cry9 and vip3A1 primers showed isolates with varied insecticidal gene combinations. Bt isolates containing cry1 genes were found to be abundant (30), followed by cry2 (9) and vip3A1 (9). Cry9 was absent in all the 60 isolates tested. Insecticidal activity of spore crystal mixtures ranged from 0 to 100% mortality. Furthermore, 12 isolates were found to be highly toxic against the larvae of diamondback moth, Plutella xylostella (L.) (Plutellidae: Lepidoptera) with 100% mortality, at 25 µg/ml in leaf disc bioassay. Conclusions The present work established the diversity of Bt isolates and confirmed the importance of continuous exploration of new Bt isolates for novel genes. Further, research needs to be carried out to unveil the hidden potential of these toxic isolates.

Pathogenicity of some local entomopathogenic fungus isolates on the cotton leafworm larvae, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

Background Pathogenicity of the entomopathogenic fungi (EPF), isolated from soil samples collected from Ordu Province, Turkey, was evaluated on the second-instar larvae of the cotton leaf worm Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) under laboratory conditions. Results Firstly, single-concentration response tests were conducted in order to determine the efficacy of the 64 isolates on S. littoralis larvae at the concentration of 1 × 108 conidia/ml. The five isolates displaying the highest mortality rates in single-concentration response tests, ORU-50, ORM-40, ORP-13, ORP-27 and ORM-48 (which included Beauveria bassiana, Metarhizium brunneum and Clonostachys rogersoniana), were subjected to concentration–response tests at the concentrations of 1 × 105–1 × 109 conidia/ml. The lowest LC50 and LC90 values were recorded at ORP-27 with 1.68 × 107 and 4.60 × 108 conidia/ml, respectively, followed by ORP-13 and ORM-40. Conclusions Accordingly, it was found that M. brunneum isolates were more effective than B. bassiana and C. rogersoniana against S. littoralis larvae. ORP-27, ORP-13 and ORM-40 of M. brunneum isolates can be a potential biological control agent used against S. littoralis larvae.

Efficacy of Thai indigenous entomopathogenic nematodes for controlling fall armyworm (Spodoptera frugiperda) (J. E. Smith)(Lepidoptera; Noctuidae)

Background Under laboratory and greenhouse conditions, the virulence of 2 isolates of Thai indigenous entomopathogenic nematodes (EPNs) in controlling the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Lepidoptera; Noctuidae), was demonstrated. Six EPNs dosages were tested against 2 larval instars of FAW under the laboratory conditions, while 2 different concentrations were tested under the greenhouse conditions. Results The results of a laboratory experiment revealed that 2 Thai indigenous EPNs isolates (Heterorhabditis indica isolate AUT 13.2 and Steinernema siamkayai isolate APL 12.3) were efficient against the FAW, 2nd and 5th larval instars. Six different nematode concentrations (50,100, 150, 200, 250 and 300 infectious juveniles (IJs) ml−1) were evaluated, and all were proven to be effective, with the mortality rate associated with concentration. Inoculated larvae in the 2nd instar was more vulnerable than that in the 5th instar. H. indica isolate AUT 13.2 was more destructive than S. siamkayai isolate APL 12.3. The greatest mortality rate of 2nd instar larvae was 83% when H. indica AUT 13.2 was applied at the concentration of 250 IJs ml−1, and 68% when the nematode S. siamkayai APL 12.3 was used at the concentration of 300 IJs ml−1. At 250 IJsml−1, the highest mortality rate of the 5th instar larvae was 45% for H. indica AUT 13.2 and 33% for S. siamkayai APL 12.3, respectively. To customize the concentration and volume of nematodes suspension evaluated in the greenhouse settings, the most sensitive stage of FAW and the optimum concentration that caused the highest mortality were used. The concentrations of both indigenous nematodes’ isolates were 20,000 and 50,000 IJsml−1 per pot, respectively, and the results showed that the mortality rates were lower than that in the laboratory. FAW mortality rate was the highest (58%) in case of the nematode H. indica isolate AUT 13.2, against (45%) in case of S. siamkayai isolate APL 12.3, at the 50,000 IJs ml−1 concentrations. Conclusions The study revealed the 2 Thai indigenous EPNs isolates (H. indica isolate AUT 13.2 and S. siamkayai isolate APL 12.3) were capable of controlling the FAW in both laboratory and greenhouse environments. The 2 Thai EPNs showed the potential to be considered as a biological control agent.