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Article

Initial Evaluation of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium robertsii, and the Entomopathogenic Nematode Heterorhabditis bacteriophora, Individually and in Combination against the Noxious Helicoverpa armigera (Lepidoptera: Noctuidae)

by
Waleed S. Alwaneen
1,
Muhammad Tahir
2,3,
Pasco B. Avery
4,
Waqas Wakil
2,5,*,
Nickolas G. Kavallieratos
6,*,
Nikoleta Eleftheriadou
6,
Maria C. Boukouvala
6,
Khawaja G. Rasool
7,
Mureed Husain
7 and
Abdulrahman S. Aldawood
7
1
Advanced Agricultural & Food Technology Institute, King Abdulaziz City for Science and Technology (KACST), Riyadh 11442, Saudi Arabia
2
Department of Entomology, University of Agriculture, Faisalabad 38040, Pakistan
3
Ministry of National Food Security and Research, Islamabad 44000, Pakistan
4
Department of Entomology and Nematology, Indian River Research and Education Center, Institute for Agricultural Sciences, University of Florida, Fort Pierce, FL 34945, USA
5
Senckenberg German Entomological Institute, D-15374 Müncheberg, Germany
6
Laboratory of Agricultural Zoology and Entomology, Faculty of Crop Science, Agricultural University of Athens, 75 Iera Odos Str., 11855 Athens, Greece
7
Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(7), 1395; https://doi.org/10.3390/agronomy14071395
Submission received: 2 May 2024 / Revised: 20 May 2024 / Accepted: 23 May 2024 / Published: 27 June 2024
(This article belongs to the Special Issue Advances and Challenges for the Management of Lepidopteran Pests—2nd Edition)
Versions Notes

Abstract

:
The Old-World bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae), is a significant threat to crops worldwide and has become resistant to traditional synthetic insecticides. The present study investigated the pathogenicity of the entomopathogenic fungi (EPF) Metarhizium robertsii (Hypocreales: Clavicipitaceae) strain WG-04 or Beauveria bassiana (Hypocreales: Cordycipitaceae) strain WG-10, and an entomopathogenic nematode (EPN) species Heterorhabditis bacteriophora against the second and fourth instar larvae of H. armigera. Both fungal species and H. bacteriophora were evaluated, singly or in combination. After 24 and 48 h post-application of the fungal spores (106 spores/mL), H. bacteriophora was introduced at a rate of 50 infective juveniles/mL, and mortality was observed at 3-, 5-, and 7-days post-treatment. Adult emergence, egg hatching, and percentage pupation were recorded. The integration of both types of biocontrol agents exhibited additive and synergistic interactions in larval stages, and enhanced mortality was recorded when EPF was used in combination with the nematodes. In the individual application of all three biocontrol agents alone, the order of efficacy was H. bacteriophora > B. bassiana > M. robertsii; however, in joint treatments, the increase in mortality and decrease in percentage egg hatching, pupation, and adult emergence was determined to be directly linked to the exposure period of the H. bacteriophora. The results of this study suggest that combining H. bacteriophora with EPF could provide a solid foundation for an economically viable method for managing H. armigera larvae in chickpea fields.

1. Introduction

Pulses have been instrumental in meeting the nutritional requirements of impoverished populations worldwide [1]. Among these, chickpea, Cicer arietinum L., is ranked second in use for food and fodder [2]. Despite its high nutritional value and growing demand, chickpea production currently stands at 14.25 Mt yearly, a figure below the global demand [3]. One of the most serious insect pests that can negatively impact chickpea production is Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) [3,4]. Helicoverpa armigera is a polyphagous and voracious cosmopolitan pest that attacks over 300 economically significant plant species, including cotton, chickpea, soybean, sunflower, groundnut, okra, tomato, pepper, tobacco, pigeon pea, maize, and sorghum [3,5,6,7].
The larvae of this lepidopteran pest exhibit a preference for consuming plant reproductive organs, including those of flowers and fruits, as well as other tender plant parts such as bolls [8]. In chickpeas, the early larval instars of H. armigera preferentially devour flowers, buds, tender twigs, and young pods, leading to 95–100% crop losses when infestation levels are high [9,10,11,12]. The early instar larvae consume foliage, while the L3 larvae transition to pod consumption, burrowing into them [13] and consuming the developing seeds [14]. Throughout its larval stage development, each larva can consume up to 40 chickpea pods [15].
The significant burden of losses on vital cash crops underscores the need for farmers to effectively manage this key pest promptly [16,17]. To manage this pest, chickpea growers commonly rely on traditional synthetic chemical insecticides [18,19]. The unwise, substandard, and repeated use of these chemical insecticides have resulted in the broad-spectrum resistance of this pest to new chemistry insecticides [20]. Reports on the failure of H. armigera management have frequently been communicated throughout Pakistan in the last 20 years due to this demonstrated resistance [20,21,22].
However, different management tactics such as resistant cultivars, cultural practices, pheromone traps, and biological and synthetic chemicals applied alone or in combination have demonstrated promising results [23]. The application of biopesticides reduces the selection pressure of insecticides and diminishes the concurrent resistance risks in target pests [24]. This combination of treatments allows for a successful integrated pest management strategy, wherein their interactions may result in additive or synergistic effects [25,26,27]. Considering this new management tactic, entomopathogenic nematodes (EPNs) and entomopathogenic fungi (EPF) have been reported to be efficient in controlling multiple insect pests [28,29]. Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) (Bb) has been proven effective for managing numerous lepidopteran insect pests including H. armigera [20,30,31]. Beauveria bassiana, as an obligate parasite and omnipresent EPF, is known to cause white muscardine disease [32], while Metarhizium robertsii J.F. Bisch., Rehner & Humber (Hypocreales: Clavicipitaceae) (Mr) is responsible for causing green muscardine disease [33]. Modes of disease spread in insects include direct contact, as well as horizontal or vertical transmission of the fungi [34,35,36].
EPN exhibits a propensity for effectively managing a wide spectrum of insect pests [37,38,39]. They are considered soil microbes targeting soil-dwelling insects but play a vital part in managing insect pests of vegetables and crops of commercial importance. These organisms belong to the Heterodabditidae and Steinernematidae families, and they target their host by utilizing a mutualistic bacterium belonging to species of the genera Photorhabdus and Xenorhabdus, respectively [40,41]. Concerning their mode of action, nematodes enter their host’s body via natural openings, where they complete two to three generations and produce infective juveniles. Following the demise of their host, the infective juveniles seek out new hosts [40].
Considering the potential of using both biocontrol agents (EPF and EPNs), they could provide an optimal alternative to conventional synthetic chemical insecticides in an integrated pest strategy for the control of field populations of H. armigera. These biocontrol agents impair the physiology, behaviour, and development of their hosts, offering an advantage against resistant species [42]. A strong, integrated mortality effect has been observed when EPF and EPNs are used in combination [43,44]. Therefore, the current study was designed to assess the interactive potential (additive, synergistic) of EPF, i.e., Bb, Mr, and an EPN, i.e., Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) (Hb), on the mortality, pupation, adult emergence, and egg eclosion of different developmental stages of H. armigera.

2. Materials and Methods

2.1. Helicoverpa armigera Culture

The trials were conducted using a colony of H. armigera initially collected from a field population in the peri-urban setting of Faisalabad, Pakistan, and maintained at the Microbial Control Laboratory, Department of Entomology, University of Agriculture, Faisalabad (UAF), in Pakistan for more than 10 years. The assays were carried out using progeny from the 25th generation, reared on the artificial diet described in Wakil et al. [45]. The second instar larvae (L2) were reared individually in 6-well plates (Fisher Scientific, Pittsburg, PA, USA) to avoid cannibalistic behaviour. Each well contained 6–8 mL of solidified diet (Southland Products Incorporated, Lake Village, AR, USA), and the larvae fed until pupation. After pupation, pupae were transferred to plastic boxes (0.3 m × 0.3 m × 0.3 m) until adult emergence [46]. After emergence, the adults fed on a diet which consisted of a 10% honey solution containing 2 g of methyl-4-hydroxybenzoate and 1 g of streptomycin sulfate [47]. Rearing conditions were maintained at 25 °C and 65% relative humidity (RH), with a 14:10 h light:dark (L:D) photoperiod, within an incubator (MIR-254, Sanyo, Tokyo, Japan) [48].

2.2. Entomopathogenic Nematode

Infective juveniles (IJs) of the EPN Hb used in the bioassay were obtained from the Microbial Control Laboratory, University of Agriculture, Faisalabad, Pakistan. The nematodes were maintained on the last instar of Galleria mellonella L. (Lepidoptera: Pyralidae) larvae following the procedure described by Kaya and Stock [49], and host insects were reared using the technique described by Van Zyl and Malan [50]. The IJs produced were stored at 14 °C in 250 mL tissue culture flasks (Fisher Scientific, USA) for less than two weeks before being used for the bioassays [51].

2.3. Entomopathogenic Fungi

The two isolates of EPF used in the study, WG-14 Bb and WG-10 Mr, were initially isolated from soil in crop fields and vegetables, respectively [46]. The original cultures were maintained on potato dextrose agar (BD, Sparks, MD, USA) slants in test tubes at 4 °C in the refrigerator (Haier, Lahore, Pakistan) at the Microbial Control Laboratory at the University of Agriculture, Faisalabad, Pakistan. Mass culturing of the individual isolates obtained was performed by inoculating glass Falcon® Petri plates (10 cm in diameter) (Fisher Scientific, Waltham, MA, USA) containing potato dextrose agar solid media with 1 × 106 conidia/mL and spread using a sterile scalpel. The plates were sealed with Parafilm (Sigma Aldrich Chemie, Taufkirchen, Germany) and placed in an incubator (MIR-254, Sanyo, Tokyo, Japan) set at 25 °C under a 14:10 h L:D photoperiod. Following an incubation period of 7–10 days, the plates were scraped using sterilized scalpels and the collected dry conidia were transferred to individual Falcon® sterile polypropylene 50 mL screw-cap tubes (Fisher Scientific, Waltham, MA, USA) comprising 30 mL of a 0.05% solution of Silwet® L-77 (bioWORLD, Dublin, OH, USA). The tubes were capped and vortexed with the addition of eight glass beads per tube for five min. The required concentration of 1 × 106 spores/mL was adjusted from the original suspension per tube using a hemocytometer (Marienfeld, Lauda-Konigshofen, Germany) under a light microscope at 400× magnification. The conidial viability was noted according to Usman et al. [52].

2.4. Bioassays

2.4.1. Effect of EPF on Helicoverpa armigera Larvae

Each of the fungal isolates (Bb and Mr) were tested at 1 × 106 spores/mL against L2 and fourth instar (L4) H. armigera larvae. There were 15 insects per treatment and the experiments were conducted 3 times, for a total of 45 insects/treatment [46,53]. The larvae were individually immersed in a 100 mL conidial suspension mixed with 0.01% Tween 80 inside a beaker (Pyrex Griffin, Sigma Aldrich Chemie, Taufkirchen, Germany) for 10 s [46]. Larvae in the control treatment were immersed individually in a 150 mL beaker containing only an aqueous solution of 0.01% Tween 80. Immediately after treatment, larvae were transported to plastic vials (ProLab Supply Co., Miramar, FL, USA) with a base radius of 120 mm × height of 80 mm, ventilation hole size of 40 mm, and a mesh pore size of 0.053 mm. Once air dried for 30 min, the larvae were transferred to new clean rearing plastic vials containing artificial diet and maintained at 25 °C, with a 65% RH under a 14:10 h L:D photoperiod in the same incubator as above [46]. The bioassay was repeated thrice independently for each set of L2 and L4 larvae of H. armigera.

2.4.2. Effect of EPN on Helicoverpa armigera Larvae

A nematode suspension of 50 IJs/mL of Hb was prepared in glass jars and the concentration was confirmed using a Neubauer-improved hemocytometer, as above. One ml of suspension was transferred to vials (ProLab Supply Co., Miramar, FL, USA) with a base radius of 120 mm × height of 80 mm, ventilation hole size of 40 mm, and a mesh pore size of 0.053 mm embedded with Whatman filter paper. The filter paper (double layer) impregnation method was employed [39,54,55]. The vials were then allowed to rest for 30 min to ensure the uniform distribution of the nematodes on the filter paper. The controls consisted of 1 mL of distilled water. A small piece (1.5 cm × 1.5 cm) of artificial diet was offered to each L2 and L4 larvae of H. armigera as a food source in the vial. A new batch of 15 larvae were individually placed in each vial. The vials were sealed with Parafilm and maintained at 25 °C, 65% RH under a 14:10 h L:D photoperiod. The larvae were agitated using a blunted needle, and if there was no observable physical response, they were deemed deceased. Mortality rates were documented at 3-, 5-, and 7-days post-incubation, when a new series of vials were prepared for each exposure. The entire bioassay was performed on three distinct occasions using fresh material to mitigate the issue of pseudo-replication.

2.4.3. Interactions between the EPF and EPN

The combined action of both EPF and EPN was tested against L2 and L4 larvae of H. armigera at different time intervals to determine their possible interaction. The joint application of the entomopathogens (EPF and EPN) occurred either concurrently or with a time gap of 24–48 h. The interactions between Mr and Bb were not assessed and are beyond the scope of this paper. Three combination treatments were implemented, each characterized by the time between the initial and the sequential application of each treatment. In the first combined treatment, larvae were individually dipped into a vial containing 1 × 106 spores/mL suspension of either Mr or Bb [46]. The treated larvae were then individually allowed to air dry for 30 min and subsequently transferred to single SPL vials lined with wet double-layer filter paper pre-treated with 50 IJs/mL of Hb. These treatments were denoted as Mr + Hb or Bb + Hb, respectively. In the second treatment, larvae were initially treated with either Mr or Bb, and after a 24 h period post-application, they were transferred to vials lined with wet double-layer filter paper treated with Hb IJs at 50 IJs/mL, as described above. These treatments were labelled Mr + Hb 24 h or Bb + Hb 24 h, respectively. In the third treatment, the fungal-treated larvae (at 1 × 106 spores/mL) were introduced into vials containing Hb IJs (at 50 IJs/mL) after 48 h from the initial application of the fungi. These treatments were designated Mr + Hb 48 h or Bb + Hb 48 h, respectively [56,57,58]. The single application of biocontrol agents as described above, plus control (0.01% Tween-80), was also included in the bioassay. All treatments were maintained at 25 °C, 65% RH under a 14:10 h L:D photoperiod. A batch of 15 larvae were individually placed in each vial. The data of mortality were observed at 3-, 5-, and 7-days post-exposure, where new sets of vials were prepared per exposure. In all treatments, an artificial diet was given to the larvae. After each mortality count, the cadavers were maintained in uncontaminated Falcon® sterile polypropylene 50 mL screw-cap tubes to monitor nematode infection symptomatology or fungal mycosis. Larvae that yielded no response to the touch with a blunt needle were recorded as dead. The egg hatching percentage, pupation, and adult emergence were also noted. The larval duration in different treatments was 16.95 ± 1.08 days to 23.36 ± 1.50 days and the pupal duration ranged between 11.23 ± 0.93 days and 15.76 ± 1.27 days. The pupation percentage and adult emergence were calculated based on the initial number of larvae used in each treatment [57,58]. However, the egg hatching percentage was calculated based on the total number of eggs hatched out of the total laid eggs in each treatment. The entire bioassay was repeated on three different occasions.

2.5. Statistical Analysis

The integration of both biocontrol agents, EPF and EPN, was tested to determine their interactions, categorized as either antagonistic, synergistic, or additive. The interactions were computed using the equation developed by Shapiro-Ilan et al. [59]. The comparison between the expected and observed percentage mortality of H. armigera served as the baseline for establishing any relationship or type of interaction. The formula for expected mortality (PE) was calculated as follows:
PE = P0 + (1 − P0) (P1) + (1 − P0) (1 − P1) (P2)
where PE represents the expected mortality of the combination, P0 is the control mortality in the control group, P1 is the individual mortality from one of the first pathogen treatments singly applied, and P2 is the individual mortality from the other pathogen singly applied. The mortality in control treatments was <4%.
The association between observed and expected mortality was tested using the χ2 test, expressed as follows:
χ2 = (L0 − LE)2/LE + (D0 − DE)2/DE
where LE indicates the expected value for alive larvae, L0 represents the observed value for alive larvae, D0 indicates the count of observed dead larvae, and DE represents the count of expected deceased larvae. Interactions were classified as additive if χ2 < 3.84, antagonistic if χ2 > 3.84 and PC < PE, and synergistic if χ2 > 3.84 and PC > PE. PC denotes the observed mortality from the combination, and PE denotes the expected mortality of the combination. Data on mortality were corrected using Abbott’s formula [60]. For data on egg eclosion, pupation, and adult emergence, a two-way analysis of variance (ANOVA) was executed using Minitab [61]. Treatment and insect larval instar were the main effects. Data were log (x + 1)-converted to normalize the variance [62]. Significant mean separation was determined using Tukey’s HSD test (5% significance level) [62].

3. Results

Effects and Interactions of EPF and EPN

The interaction of Bb and Mr with Hb was most virulent, according to the highest mortality percentage of H. armigera, in treatments with sequential applications of both entomopathogens after 48 h. Complete mortality (100%) was observed in the L2 larvae of H. armigera when treated with Bb + Hb 48 h at 7 days post-exposure (Table 1), while the maximum percentage mortality for the L4 larvae was 89.69% for the same treatment (Table 2).
The synergistic interactions were more profound against L2 larvae of H. armigera (Table 1), whereas in the case of L4 larvae, more additive interactions were recorded, except in treatments where Bb + Hb was applied at a 48-h interval (Table 2).
The pattern indicated a synergistic effect in the sequential application at 24 h apart for Bb and Hb after 7 days of incubation. In contrast, additive effects were demonstrated for the Mr and Hb combined treatment at a 24 h interval. At the 48-h interval, a synergistic interaction was observed with the Bb + Hb treatments at 3 and 7 days post-application, whereas synergism was specifically observed at 7 days post-application for the Mr + Hb combination treatment (Table 2).
In the factorial analysis, the main effects for egg eclosion, pupation, and adult emergence were found to be significant, whereas their interaction effects were not significant, apart from pupation (Table 3).
An inverse relationship was revealed among the egg eclosion, pupation, and adult emergence of surviving H. armigera larvae and combined treatments of the microbial control agents (Table 4).

4. Discussion

Food security and sustainability in agriculture depend upon the availability of protein-enriched legumes like chickpea [3]. Helicoverpa armigera is one of the most devastating pests of chickpea. Therefore, the mitigation of H. armigera damage by using alternative integrated management strategies can potentially increase chickpea crops’ production, minimize broad-spectrum synthetic insecticide usage, and ensure sustainable nutritional food security [22].
In the present study, the integrated impact of the EPF Bb and Mr with the EPN Hb against the larvae of H. armigera was evaluated. The findings revealed that both EPFs (Bb or Mr) can efficiently manage the pest’s larval stages when applied either simultaneously or sequentially in combination with Hb at 24 or 48 h after the fungal treatment. However, the application of each of the three agents alone did not demonstrate significant effectiveness compared to the control group. Additive and/or synergistic interactions were recorded for joint applications of Hb at 24 and 48 h after the application of Mr and Bb. The interaction type (additive, synergistic, or antagonistic) strongly depends on the target insect pest, control agents, application time, and infection dose [63,64]. When two biocontrol organisms are employed separately in a combined infection, their interaction may vary. If their combined effect is simply the sum of their individual effects, the interaction is considered additive. However, if the combined effect differs significantly from the additive effect, the interaction is categorized as either synergistic or antagonistic [64]. The same trend was exhibited in the results of the present study.
An additive or synergistic interaction on the mortality of H. armigera was inflicted when EPF and EPN were inoculated simultaneously. Comparable positive effects, either additive or synergistic, resulting from interactions between EPF and EPN have been documented by other researchers [44,63,64,65,66,67,68,69]. For instance, Mr combined with the EPN Heterorhabditis indica Poinar, Karunakar & David (Rhabditida: Heterorhabditidae) exhibited additive effects in insect mortality when simultaneously applied against larvae of Curculio caryae Horn (Coleoptera: Curculionidae) [59]. Similarly, additive interactions were identified for four EPF and two EPN species against Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) across all combinations of EPF + EPN tested [52]. Although the mechanism for these positive interactions of combined applications is still unclear, it can be hypothesized that one of the agents (EPF or EPN) acts as a stressor [70]. This stressor disrupts the homeostatic balance of the target insect, weakening the host and rendering it more susceptible to infection by the pathogen(s). Consequently, this increased vulnerability contributes to heightened mortality among target insects [71].
Furthermore, it is noteworthy that weakened insects exhibit heightened respiration rates, thereby releasing more CO2, which attracts more infective juvenile EPNs and promotes parasitic activity [65,72]. For example, Paenibacillus popilliae Dutky (Bacilliales: Paenibacillaceae), a soil-dwelling Gram-positive bacterium, when administered to scarab larvae, functioned as a stressor for EPN infection, causing increased larval mortality [73,74]. According to Steinhaus [70], stressed insects are particularly susceptible to infection by a pathogen, therefore increasing insect mortality or improving the rate of kill. This phenomenon also contributes to the promotion of either additive or synergistic effects in combined treatments [70]. The synergistic interaction observed by Correa-Cuadros et al. [64] between Hb HNI0100 in combination with Mr 9236 or Bb 9205 against Plutella xylostella (L.) (Lepidoptera: Plutellidae) further supports this EPF + EPN combination against different insect species. Sáenz-Aponte et al. [68] interpreted the decrease in broccoli plant damage percentage as being due to the combined application of Hb HNI0100 with Mr 9236 and Bb 9205 against P. xylostella larvae, compared to the controls. In addition, the authors suggested that the delayed application of nematodes up to 6 days after the application of EPF resulted in a stronger synergistic interaction. In the present study, enhanced efficacy, along with both additive and synergistic interactions, was observed after a delay of 24 or 48 h before the application of Hb.
In contrast, Shapiro-Ilan et al. [59] identified antagonistic interactions between EPNs and Cordyceps fumosorosea (Wize) Kepler, B. Shrestha & Spatafora (Hypocreales: Cordycipitaceae). This disparity in results could stem from pathogen interactions occurring during or before infection. In the case of the bacterium Serratia marcescens Bizio (Enterobacterales: Yersiniaceae) found in the EPNs and C. fumosorosea, it is plausible that C. fumosorosea may be infectious to different species of EPNs, leading to a decrease in the infectivity of nematodes or their elimination before causing infection. Antagonism between the two agents might arise from toxins produced by microbes before the manifestation of the disease in the host [59]. In the present study, no antagonistic effects were observed for the combined EPF and EPN against H. armigera larvae. Nevertheless, Hb demonstrated higher virulence compared to the EPF when applied as a single treatment. This trend has been observed where EPNs have previously exhibited elevated virulence against C. caryae and Diatraea saccharalis (F.) (Lepidoptera: Crambidae) compared to the EPF applied alone [59,67].
Wakil et al. [44] reported an elevated rate of adult emergence and egg hatching along with a decrease in pupation during the developmental duration of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), following the combined application of Bb and Hb. Also, Marzban et al. [58] observed that the combination of Cry1Ac + HaCPV had a significant effect on the first instar of H. armigera larvae, resulting in reduced pupal weight and pupation, and a prolonged developmental period. Nguyen et al. [75], who studied the susceptibility, pupation, and adult emergence of H. armigera L3 larvae when treated with Mr, Bb, and C. fumosorosea, demonstrated prolonged pupation and a negative impact on adult performance, likely affecting adult emergence and viability. This behavioural response of insects after being infected by different entomopathogens extends beyond the lepidopteran species. As an illustration, the impact on Blattella germanica L. (Blattodea: Ectobiidae) revealed significant differences in egg production, hatching percentage, and adult emergence when subjected to low doses of Mr [76]. These behavioural phenomena could be attributed to the unfavourable physiological alterations in females, leading to prolonged immature phases and a diminished adult lifespan [77,78,79]. However, determining the causes of the behavioural responses of arthropods after being infected with entomopathogens was not the focus of this study; thus, these hypotheses would require further study.

5. Conclusions

In conclusion, the synergistic and additive effects observed between the tested EPF and EPN present a promising and innovative alternative approach for effectively managing H. armigera. Consequently, integrating both biological control agents into an integrated pest management program is recommended for the control of this lepidopteran pest. Subsequent studies conducted under field settings will be necessary to validate the outcomes of this laboratory-based research.

Author Contributions

Conceptualization, W.W.; methodology, W.W.; software, W.S.A., M.T., and W.W.; validation, W.S.A., M.T., P.B.A., W.W., N.G.K., N.E., M.C.B., K.G.R., M.H., and A.S.A.; formal analysis, W.S.A., M.T., P.B.A., W.W., N.G.K., N.E., M.C.B., K.G.R., M.H., and A.S.A.; investigation, W.S.A., M.T., P.B.A., W.W., N.G.K., N.E., M.C.B., K.G.R., M.H., and A.S.A.; resources, W.W.; data curation, M.T. and W.W.; writing—original draft preparation, W.S.A., M.T., P.B.A., W.W., N.G.K., N.E., M.C.B., K.G.R., M.H., and A.S.A.; writing—review and editing, W.S.A., M.T., P.B.A., W.W., N.G.K., N.E., M.C.B., K.G.R., M.H., and A.S.A.; visualization, W.W.; supervision, W.W.; project administration, W.W. and N.G.K.; funding acquisition, W.W. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported (2AV1-257) by the Higher Education Commission, Islamabad, Pakistan.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The KSU authors are thankful to Researchers Supporting (Project RSPD2024R721), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Mortality (mean %) of L2 larvae of H. armigera treated with B. bassiana (Bb), M. robertsii (Mr), and H. bacteriophora (Hb) in combined treatments at different time intervals. Both B. bassiana and M. robertsii were applied at 1 × 106 spores/mL and H. bacteriophora at 50 IJs/mL.
Table 1. Mortality (mean %) of L2 larvae of H. armigera treated with B. bassiana (Bb), M. robertsii (Mr), and H. bacteriophora (Hb) in combined treatments at different time intervals. Both B. bassiana and M. robertsii were applied at 1 × 106 spores/mL and H. bacteriophora at 50 IJs/mL.
TreatmentInterval a (h)Days bObserved Mortality (%)Expected Mortality (%)χ2Interaction
Mr + Hb0324.4820.400.68+ c
0537.7529.341.87+
0753.0640.962.75+
Bb + Hb0329.8923.031.57+
0550.5139.502.40+
0761.8545.634.25++ d
Mr + Hb24329.8923.901.20+
24545.3634.212.73+
24763.9147.524.20++
Bb + Hb24339.1729.152.56+
24555.6740.714.01++
24774.2254.085.46++
Mr + Hb48345.3634.212.73+
48563.9147.524.20++
48786.5963.146.35++
Bb + Hb48356.7040.714.50++
48576.2854.086.46++
487100.0069.289.43++
a Interval between the application of EPF and EPN. b Days after fungal application. c +: Additive. d ++: Synergistic.
Table 2. Mortality (mean %) of L4 larvae of H. armigera treated with B. bassiana (Bb), M. robertsii (Mr), and H. bacteriophora (Hb) in combined treatments at different time intervals. Both B. bassiana and M. robertsii were applied at 1 × 106 spores/mL and H. bacteriophora at 50 IJs/mL.
Table 2. Mortality (mean %) of L4 larvae of H. armigera treated with B. bassiana (Bb), M. robertsii (Mr), and H. bacteriophora (Hb) in combined treatments at different time intervals. Both B. bassiana and M. robertsii were applied at 1 × 106 spores/mL and H. bacteriophora at 50 IJs/mL.
TreatmentInterval a (h)Days bObserved Mortality (%)Expected Mortality (%)χ2Interaction
Mr + Hb0317.3415.750.14+ c
0528.5723.151.02+
0741.8333.621.61+
Bb + Hb0322.6818.470.78+
0541.2332.841.70+
0749.4836.883.20+
Mr + Hb24322.6818.470.78+
24534.0227.421.27+
24753.6039.863.52+
Bb + Hb24328.8622.091.58+
24543.2931.693.10+
24762.8846.114.47++ d
Mr + Hb48336.0827.422.07+
48553.6039.863.52+
48776.2856.015.38++
Bb + Hb48344.3231.693.60+
48564.9446.115.46++
48789.6963.347.73++
a Interval between the application of EPFs and EPNs. b Days after fungal application. c +: Additive. d ++: Synergistic.
Table 3. ANOVA parameters for pupation, adult emergence, and egg eclosion of H. armigera treated with B. bassiana, M. robertsii, and H. bacteriophora.
Table 3. ANOVA parameters for pupation, adult emergence, and egg eclosion of H. armigera treated with B. bassiana, M. robertsii, and H. bacteriophora.
PupationAdult EmergenceEgg Eclosion
EffectdfFpFpFp
Treatment9121.11<0.01290.32≤0.01232.18<0.01
Instar1229.13<0.0119.61≤0.0116.97<0.01
Treatment × Instar997.48<0.010.460.900.520.85
Error152
Total179
Table 4. Mean (% ± SE) pupation, adult emergence, and egg eclosion of L2 and L4 H. armigera larvae treated with B. bassiana (Bb), M. robertsii (Mr), and H. bacteriophora (Hb) in single and combined treatments at different time intervals. Both B. bassiana and M. robertsii were applied at 1 × 106 spores/mL and H. bacteriophora at 50 IJs/mL. Mean values followed by different letters within each column per L2 or L4 are significantly different (Tukey HSD test, p = 0.05).
Table 4. Mean (% ± SE) pupation, adult emergence, and egg eclosion of L2 and L4 H. armigera larvae treated with B. bassiana (Bb), M. robertsii (Mr), and H. bacteriophora (Hb) in single and combined treatments at different time intervals. Both B. bassiana and M. robertsii were applied at 1 × 106 spores/mL and H. bacteriophora at 50 IJs/mL. Mean values followed by different letters within each column per L2 or L4 are significantly different (Tukey HSD test, p = 0.05).
L2 L4
TreatmentsTime Interval (h)Pupation (%)Adult Emergence (%)Egg Eclosion (%)Pupation (%)Adult Emergence (%)Egg Eclosion (%)
Mr 73.34 ± 3.31 b65.57 ± 2.93 b58.85 ± 3.51 b80.54 ± 3.29 b72.23 ± 2.77 b65.51 ± 2.93 b
Bb 64.46 ± 2.93 bc57.74 ± 2.77 bc51.14 ± 2.04 bc71.12 ± 3.54 bc64.45 ± 2.93 bc58.83 ± 3.09 bc
Hb 57.73 ± 2.23 c51.62 ± 2.35 c46.68 ± 2.33 c63.36 ± 2.35 c58.84 ± 2.23 c53.37 ± 2.63 c
Mr + Hb040.02 ± 1.63 d34.41 ± 1.93 d29.43 ± 1.69 d46.61 ± 2.88 d42.28 ± 3.14 d37.74 ± 2.21 d
Bb + Hb025.58 ± 1.93 e20.53 ± 1.25 e16.61 ± 1.16 e29.48 ± 1.42 e24.40 ± 1.35 e21.16 ± 1.13 e
Mr + Hb2419.41 ± 1.79 ef15.58 ± 1.13 ef12.25 ± 1.02 e24.43 ± 1.93 ef21.11 ± 2.05 ef17.70 ± 1.46 e
Bb + Hb2415.54 ± 1.75 ef12.21 ± 1.09 ef10.57 ± 1.00 ef18.82 ± 1.21 efg15.53 ± 1.22 efg12.26 ± 1.22 ef
Mr + Hb4811.67 ± 1.17 f9.55 ± 0.89 fg7.72 ± 0.78 ef13.36 ± 1.06 fg10.57 ± 1.11 fg9.47 ± 0.93 ef
Bb + Hb480.00 ± 0.00 g0.00 ± 0.00 g0.00 ± 0.00 f6.69 ± 0.66 g4.45 ± 0.25 g2.25 ± 0.13 f
Control 93.35 ± 2.35 a89.34 ± 3.79 a83.36 ± 3.35 a95.54 ± 1.75 a91.11 ± 2.13 a85.53 ± 3.76 a
df 9, 899, 899, 899, 899, 899, 89
F 150.0178.0120.098.0122.0117.0
p <0.01<0.01<0.01<0.01<0.01<0.01
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MDPI and ACS Style

Alwaneen, W.S.; Tahir, M.; Avery, P.B.; Wakil, W.; Kavallieratos, N.G.; Eleftheriadou, N.; Boukouvala, M.C.; Rasool, K.G.; Husain, M.; Aldawood, A.S. Initial Evaluation of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium robertsii, and the Entomopathogenic Nematode Heterorhabditis bacteriophora, Individually and in Combination against the Noxious Helicoverpa armigera (Lepidoptera: Noctuidae). Agronomy 2024, 14, 1395. https://doi.org/10.3390/agronomy14071395

AMA Style

Alwaneen WS, Tahir M, Avery PB, Wakil W, Kavallieratos NG, Eleftheriadou N, Boukouvala MC, Rasool KG, Husain M, Aldawood AS. Initial Evaluation of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium robertsii, and the Entomopathogenic Nematode Heterorhabditis bacteriophora, Individually and in Combination against the Noxious Helicoverpa armigera (Lepidoptera: Noctuidae). Agronomy. 2024; 14(7):1395. https://doi.org/10.3390/agronomy14071395

Chicago/Turabian Style

Alwaneen, Waleed S., Muhammad Tahir, Pasco B. Avery, Waqas Wakil, Nickolas G. Kavallieratos, Nikoleta Eleftheriadou, Maria C. Boukouvala, Khawaja G. Rasool, Mureed Husain, and Abdulrahman S. Aldawood. 2024. "Initial Evaluation of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium robertsii, and the Entomopathogenic Nematode Heterorhabditis bacteriophora, Individually and in Combination against the Noxious Helicoverpa armigera (Lepidoptera: Noctuidae)" Agronomy 14, no. 7: 1395. https://doi.org/10.3390/agronomy14071395

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