Aspergillus flavus as an Entomopathogen Infecting Diaphania indica and Control Efficacy Across Different Developmental Stages

Aspergillus flavus as an Entomopathogen Infecting Diaphania indica and Control Efficacy Across Different Developmental Stages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Aspergillus flavus as an Entomopathogen Infecting Diaphania indica and Control Efficacy Across Different Developmental Stages mofeed Abdelhamed Aboelhassan Askar, Chen Chen, Ali Borham, Xijun Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4296110/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Diaphania indica (Lepidoptera: Crambidae) is one of the most important pests infesting many cucurbitaceous vegetables. During the rearing of insect eggs, we observed a fungal infection of these insect eggs. The fungus produces aflatoxins which are considered secondary polyketide metabolites, which cause the death of pests. Therefore, this work aimed to isolate and identify this fungus by amplifying the internal transcribed spacer (ITS) region of the rDNA, as well as evaluating the efficiency of this fungus in control. Aspergillus flavus, 'PP125556,' showcased robust pathogenicity against a range of D. indica pests. The results showed that colonies of 'PP125556' cultivated on potato dextrose agar (PDA) exhibited distinctive morphological characteristics, transitioning from pristine white to verdant green. Bioassays demonstrated concentration-dependent mortality rates of D. indica larvae and adults when exposed to varying concentrations of 'PP125556' conidia, with the highest concentration (1x10 9 conidia/ml) inducing significant death with the highest mortality (53.06% for eggs, 70.57% for larvae, and 86.65% for adults). Furthermore, examination under a stereomicroscope revealed conspicuous external symptoms in infected larvae, including reduced mobility, darkened body pigmentation, and the emergence of white hyphae, indicative of mortality. Additionally, infected eggs exhibited inhibited hatching and the emergence of green hyphae, while infected adults displayed mortality and white hyphae colonization, underscoring the potent biocontrol efficacy of A. flavus 'PP125556' against D. indica across diverse developmental stages. Biological control Diaphania indica Aspergillus flavus molecular identification fungal PCR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Globally, cucurbitaceous vegetables hold a significant position in both production and consumption, constituting a substantial portion of the vegetable crop market. The Cucurbitaceae family encompasses a diverse range of vegetable crops cultivated for both culinary and medicinal purposes. These crops yield leaves, flowers, fruit, seeds, and roots, each possessing various pharmacological and pharmaceutical properties. Across culinary traditions, cucurbits feature prominently, serving as salad staples like cucumber, gherkin, and long melon, or finding utility in pickled forms such as cucumber, gherkin, and bitter gourd. Additionally, certain cucurbits like muskmelon and watermelon are enjoyed as dessert fruits, while others like ash gourd are transformed into candied or preserved delicacies. Notably, cucumber seeds are valued for their oil, which is recognized for its beneficial effects on brain and body health [ 1 ]. Originating in Asia, cucumbers have a rich history spanning over 3000 years, introduced to China around 100 B.C. and later reaching France by the 9th century [ 1 , 2 ]. Hosseinzade et al. (2014) corroborated the infestation of Cucurbitaceae by Diaphania species, noting that the larvae target young fruit after consuming the leaves entirely. Cucumis sativus L. , Sicyos angulatus L. , Luffa cylindrica L. , and various gourds including Momordica charantia L . and Citrullus lanatus Thunb are among the preferred hosts for Diaphania species. larvae. Additionally, [ 3 ] highlighted the prolific reproductive nature of Diaphania indica populations, particularly in regions such as Hainan Province, China, where multiple generations per year, possibly exceeding ten, are observed. Active D. indica populations persist throughout the year, peaking between April and September. Larval feeding activities not only lead to leaf skeletonization but also pose significant threats to flower and fruit development, often rendering them unmarketable due to secondary pathogen infections. Silk webs formed over entry holes further obstruct natural enemies' access, further exacerbating fruit-feeding damage. The importance of entomopathogenic fungi as alternative pest control agents continues to escalate, offering viable solutions to pressing agricultural challenges. Previous research has extensively utilized four bio-pesticides Beauveria bassiana , Nomuraea rileyi, Bacillus thuringiensis , and Helicoverpa armigera Nucleopolyhedrovirus (HaNPV) against Diaphania indica , demonstrating their effectiveness and ability to induce high mortality rates [ 4 ]. [ 3 ] underscored the limited success of biological control agents, highlighting the entomopathogenic nematode Steinernema carpocapsae as a promising candidate. [ 5 ] evaluated various integrated pest management (IPM) strategies, revealing D. stantoni , N. rileyi, and B. bassiana as effective controls compared to alternative approaches. Confirming these findings, [ 6 ] emphasized the efficacy of different IPM applications utilizing Dolichogenidea stantoni, Trichogramma chilonis, N. rileyi, B. bassiana, Metarhizium anisopliae , and B. thuringiensis for managing D. indica in bitter gourd, providing substantial control over the pest population. These mycopesticides primarily utilize propagules such as conidia, blastospores, and hyphae, offering immediate pest eradication and triggering secondary infection by dispersing mycotic spores horizontally from cadavers. Notably, wettable powder-based biopesticides have harnessed these fungi, further emphasizing their potential in sustainable pest management strategies [ 7 ]. Aspergillus flavus is an important entomopathogenic fungi [ 8 ] observed that A. flavus impacts Spodoptera litura by directly influencing its immune system, leading to a reduction in immune functionality. A. flavus specifically targets third and fourth instar larvae of S. litura. According to preliminary findings, the cosmopolitan saprophytic fungus known as A. flavus shows potential as a promising agent for controlling aphids [ 9 , 10 ]. Initial laboratory experiments revealed that A. flavus can effectively target aphids, with a mortality rate observed at concentrations of 1.23 × 10 3 spores/mL (LC50) and 1.34 × 10 7 spores/mL (LC90) [ 11 ]. Additionally, in a separate investigation focusing on cabbage and wheat, significant susceptibility to fungal biocontrol was observed, with 33% of cabbage aphids and 37% of wheat aphids affected, and A. flavus demonstrat notable efficacy among the pathogens tested [ 12 ]. Nonetheless, the precise mechanism through which A. flavus regulates aphid populations, as well as the specific active components involved, remains largely unexplored. 2. Materials and Methods 2.1. Isolation and Identification of the Strain In August 2023, we isolated a strain from infected Diaphania indica eggs collected from Yangzhou, Jiangsu Province, China, and inoculated it on potato dextrose agar medium (PDA) using the spread plate method [ 13 , 14 ]. Each gradient was repeated three times and cultured for seven days at 27 ̊C. Purification was repeated until a single colony was obtained [15]. For the strain, two genes were used for molecular identification according to [ 16 ]. PCR amplification was performed: (the forward primer and reverse primer were synthesized by Tsingke Biotech Technology, including the internal transcribed spacer (ITS) gene with primers ITS1 (5′-TCCGTAGGTGAACCTGCG-3′) and ITS4 (5′-TCCTCCGC TTATTGATATGC-3′), as well as a segment of the calmodulin gene (CaM) with primers cmd5 (5′-CCGAGTACAAGGAGGCCTTC-3′) and cmd6 (5′-CCGATAG AGGTCATAACGTGG-3′) (submitted separately to GenBank with accession numbers PP125556 for ITS and PP464989 for CaM ). The PCR protocol was conducted as described by White and Bruns [ 17 ]. Sequences were aligned and compared to existing sequence data in the GenBank database using the Basic Local Alignment Search Tool (NCBI BLAST) [ 18 ]. 2.2. Microscopy Identification The mycelium obtained from the Petri dishes was cultured on microscope glass slides covered with a thin layer of Sabouraud agar, following a method previously described by [ 19 ]. The cultures were then incubated at 27 ̊ C for three days. Afterwards, the fungal morphology and the cultures on the microscope glass slides were identified using microscopy. 2.3. Phylogenetic Analysis ITS and CaM sequence data of the isolate were subjected to phylogenetic analysis by comparison with registered sequences in the GenBank database. The ITS and CaM sequences were concatenated by using PhyloSuite software and the tree was built by MEGA using the NJ method [ 20 ]. Before the phylogenetic tree was built using MEGA 7.0 software, which provided the topology and length of the branches, ITS and CaM sequences were aligned, and superfluous sections were deleted [ 21 ]. 2.4. Mass Rearing of Test Insects The larvae and pupae of D. indica were removed from the cucumber plants that were infected to raise the initial culture. They were raised individually in jars (20.0 cm in height and 15.0 cm in diameter), which were regularly supplied with fresh fruit and leaves. The excreta from the half-consumed fruit and leaves were removed. Twenty pairs of newly emerged male and female adults were placed in ten mating jars, each measuring 20.0 cm in height and 15.0 cm in diameter. Fresh cucumber leaves (for laying eggs) and a cotton swab dipped in a 10% diluted honey solution (for feeding the adults) were placed inside these jars. Gravid females produced second-generation (eggs, larvae, and adults), which were used for additional treatments and sprayed with fungus spore solutions. The temperature and relative humidity during the maintenance of culture ranged from 26 ± 1°C to 27 ± 1°C and 69 ± 1 to 70 ± 1%, respectively, according to [ 22 ]. 2.5. Activity of Aspergillus flavus against Diaphania indica Under Laboratory Conditions To prepare the spore suspension, incubation plates were washed with sterile water on a sterile operating table. The spores were collected in a conical flask with glass beads, shaken thoroughly, and scattered. A single spore suspension was obtained via filtration with a cotton-stuffed syringe. Concentrations were adjusted to concentration to 10 7 , 10 8 , and 10 9 spores/mL. Finally, 0.1% tween-20 was added, and the suspension was refrigerated (4ºC). For treatment, three groups of pests were sprayed with solutions containing 10 7 , 10 8 , and 10 9 spores/mL for each treatment. An additional control group was sprayed without fungal spores for the same timeframe and with the same statistical design as for the treatments. Notably, the control group included four replicas. We did these treatments for eggs, larvae, and adults.For the egg stage, larvae, and adults of D. indica 60 eggs were counted, 20 larvae ”3rd instar” and 15 healthy adults per four replicates for each concentration were transferred to the treated cucumber leaf. A single dose of 2 ml of suspension of each concentration was sprayed on the surface of the leaf disc and the replicates using a hand spray atomizer at a distance of 25–30 cm. After the applications, we registered the death rate daily. The mortality percentage of mites was calculated and corrected according to Abbott's formula [ 23 ]. Also, the reduction percentage of hatchability was calculated according to [ 24 ]. 2.6. Statistical Analyses For statistical analysis, exact numbers were used in order to remove any analytical mistakes. The single-factor analysis of variance was carried out using SPSS 26.0, and the mean ± standard deviation was the output. Duncan's technique was applied to several comparisons. The p < 0.05 criterion was used to evaluate statistical significance. 3. Results 3.1. Isolation and Identification of the Strain Following 2–3 days of cultivation on potato dextrose agar (PDA), the strain manifested loose, flat colonies with regular edges, spanning 2–3 cm in diameter. Initially, the colonies exhibited a pristine white hue, gradually transitioning to a verdant green at their centers, characterized by a profusion of green spores (Fig. 1 A,E). Notably, the nutritional hyphae displayed septation, while a segment of the aerial hyphae extended into elongated, rough conidiophores, culminating in nearly spherical apical sacs (Fig. 1 C). Moreover, the colony surface bore numerous small peduncles, each adorned with clusters of coarse-surfaced spherical conidia (Fig. 1 D). Conidial heads that were yellow-green when young, shifted to olive-green in age (Fig. 1 B). DNA fragment sequencing unveiled gene lengths of 559 bp for ITS and 481 bp for CaM in the strain. The DNA sequences were duly submitted to GenBank, acquiring accession numbers (PP125556 for ITS and PP464989 for CaM ). BLASTN comparison against GenBank entries confirmed similarity with the reported A. flavus strains (Supplemental information). Phylogenetic analysis positioned the strain close to the A. flavus strains (Fig. 2 ), corroborating our morphological identification of the strain as A. flavus . 3.2. Biocontrol Efficacy of A. flavus ‘PP125556’ and Dose–Response Bioassay The outcomes reveal the mortality percentages of D. indica subsequent to the application of varying concentrations of conidia/ml, as depicted in (Table 1 ). Significantly, the results unveiled a marked influence on mortality rates relative to the concentration of conidia/ml. Notably, T1 (1 x 10⁹ conidia/ml) exhibited the highest mortality rates, recording 53.06% ± 4.71 for eggs, 70.57% ± 15.81 for larvae, and 86.65% ± 6.42 for adults. These values were notably distinct from those of the other treatments (P < 0.05). Moreover, T2 (1 x 10⁸ conidia/ml) and T3 (1 x 10⁷ conidia/ml) demonstrated considerable mortality rates, with 41.22% ± 8.67 and 30.20% ± 4.32 for eggs, 56.22% ± 16.29 and 39.47% ± 4.58 for larvae, and 69.00% ± 3.21 and 41.72% ± 3.71 for adults, respectively. Conversely, the control treatment (T4) yielded the lowest mortality rates, registering 1.25% ± 1.59 for eggs, 3.75% ± 2.50 for larvae, and 3.34% ± 3.85 for adults. These values exhibited significant deviations from those of the treated groups (P < 0.05). Thus, the findings underscore a concentration-dependent effect on D. indica mortality rates, with higher concentrations correlating to increased mortality. The study underscores the importance of dosage precision in entomopathogenic fungal applications for effective pest management. Notably, treatments with higher concentrations of conidia/ml exhibited pronounced mortality effects on D. indica across all developmental stages, indicative of the potential for targeted control strategies. These findings contribute valuable insights into optimizing fungal biopesticide formulations and deployment protocols for enhanced efficacy in integrated pest management programs. Table 1 Effect of different concentrations of Aspergillus flavus on Diaphania indica mortality Conc. (conidia/ml) Mortality % of Diaphania indica After Applications Egg Larvae Adult T1(1 x 10 9 ) 53.06 cd 70.57 b 86.65 a ± 4.71 ± 15.81 ± 6.42 T2(1 x 10 8 ) 41.22 de 56.22 c 69.00 b ± 8.67 ± 16.29 ± 3.21 T3(1 x 10 7 ) 30.20 e 39.47 e 41.72 de ± 4.32 ± 4.58 ± 3.71 T4 (Control) 1.25 f 3.75 f 3.34 f ± 1.59 ± 2.50 ± 3.85 Values are means of replicates ± standard deviation a−h means within a column with different letters are significantly different (P < 0.05) These findings highlight the intricate interplay between temporal dynamics and treatment modalities in shaping mortality patterns within the targeted pest populations, thus providing valuable insights for optimizing pest management strategies Figs. 1 and 2 illustrate the temporal progression of mortality rates for each treatment (T1, T2, T3, and T4) administered to the specified larval and adult stages, respectively. Within Treatment T1, there is a discernible and consistent escalation in mortality percentage observed throughout the duration of the study, indicative of a progressive impact on the targeted larval and adult populations. Similarly, Treatment T2 manifests an ascending trend in mortality rates over the observation period, albeit potentially at a different pace compared to T1, reflecting the temporal dependency of treatment efficacy. Treatment T3 also showcases an increase in mortality percentages over time, although the rate of ascent may be relatively subdued in comparison to T1 and T2. In contrast, Treatment T4, functioning as the control, exhibits relatively static or minimal mortality percentages across the monitored duration, signalling negligible effects on pest mortality over time. 3.3. External Symptoms of D. indica Infected with A. flavus ‘PP125556’ Examination of eggs under a stereomicroscope three days post-infection revealed no hatching, while after two days, green hyphae were observed emerging from the eggs (Fig. 3 A). Furthermore, Examination under a stereomicroscope unveiled negligible changes in the mobtility or physical attributes of D. indica larvae 24 hours post-infection (Fig. 3 B). Nonetheless, a notable reduction in D. indica mobility was discernible after 48 hours of infection, concomitant with darkened body pigmentation and the emergence of black intersegmental folds (Fig. 3 C). Following 72 hours of infection, white hyphae proliferated on the head, abdomen, and thorax of D. indica (Fig. 3 D), indicating their demise [ 25 ]. Additionally, their bodies displayed signs of desiccation and stiffness. Upon inspecting adults, no significant alterations in mobility were noted after 24 hours, but after 48 hours we started to record mortalities; however, when placed in moist cotton, evidence of infection surfaced, characterized by white hyphae colonization on their head, abdomen, and thorax, subsequently transitioning to green (Fig. 3 E). 4. Discussion This study delved into the isolation of A. flavus from D. indica eggs, aiming to understand its role in biocontrol efficacy. By meticulously characterizing A. flavus from these eggs, we sought to elucidate its potential in combatting D. indica infestations. Through comprehensive analyses, we aim to uncover the mechanisms underlying A. flavus interaction with D. indica populations, contributing to the development of sustainable strategies for pest management. The insights gained from this research hold promise for the formulation of targeted and environmentally friendly approaches to controlling D. indica , benefiting agricultural and ecological systems while advancing our understanding of fungal biocontrol agents. Entomopathogenic fungi (EPF) strains serve as indispensable assets in combatting insect pests, handling yield losses, and averting quality deterioration in agricultural and forestry domains. These fungi, heralded as valuable alternatives to traditional synthetic insecticides, are widely deployed to efficiently manage pests within agroecosystems [ 26 ]Notably, compared to other microbial adversaries, EPFs offer multifaceted advantages in pest control applications [ 27 ]. Despite insects' intricate arrays of defense mechanisms against infective agents, pathogens have undergone coevolution to circumvent these defenses, emphasizing the dynamic interplay in host-pathogen interactions [ 26 ] While research predominantly focuses on EPF resources like Beauveria species, Metarhizium species , and Lecanicillium species. [ 28 ], further exploration of diverse EPFs is imperative for bolstering pest management efficacy. Enter the diverse realm of Aspergillus species, renowned for their biological versatility and multifaceted utility. Notably, certain Aspergillus species, including A. flavus, A. nomius, A. fijiensis , and A. oryzae , have demonstrated remarkable efficacy against a spectrum of insect pests including Locusta migratoria, Spodoptera litura, Diaphorina citri , and Dolichoderus thoracicus [ 29 – 31 ]. Our study showed the revelation of the isolation and identification of a potent A. flavus strain from infected D. indica eggs, wielding formidable pathogenicity against agricultural insect pests. The A. flavus strain emerges as a promising candidate for broader spectrum EPF deployment in D. indica control strategies. D. indica infected via spore dissemination may act as reservoirs for perpetuating infection and facilitating further incursions into additional D. indica populations [ 32 ]The present study confirms that the fungal isolate of A. flavus is a potential biocontrol agent for the management of D. indica . The mortalities of these pests were found to be proportional to the conidial concentrations used in the bioassay involving the fungal isolate. The efficacy of A. flavus was previously studied against mango seed weevil, Sternochetus mangiferae (Coleoptera: Curculionidae) , producing mortality rates of 80% at a conidial concentration of 6.8×10 7 conidia/mL. Aspergillus species have been reported to kill the tropical locust Zonocerus variegatus and cause infection among many insect populations [ 33 ]. However, these Aspergillus species are unknown whether it is host-specific. Previous studies have reported the effectiveness of A. flavus against a wide range of insects [ 11 , 34 ]. A. flavus was also tested against Galleria mellonella , killing 100% of insects after 48 h when injected with 3 × 103 conidia/mL of the fungus. In contrast, A. fumigatus and A. nidulans lacked parasitic attributes [ 35 ]. In recent years, entomopathogenic fungi have gained attention as potential biocontrol agents, yet their safety and mode of action remain unclear. For application of A. flavus to pest management, two problems should be considered. A. flavus could produce a variety of mycotoxins, such as aflatoxins, cyclopiazonic acid and 3-nitropropionic acid, that have strong carcinogenic, teratogenic, and organ necrosis properties that threaten the food safety of agricultural products [ 36 , 37 ]. On the other hand, this kind of fungus may cause human invasive aspergillosis or allergenic problems [ 38 , 39 ]. [ 8 ] explored the toxicity of A. flavus on S. litura , analyzing its effects on antioxidant and immune defenses. It showed increased antioxidant activity and negative impacts on haemocytes. However, genotoxicity assessments in rats showed no significant effects, suggesting further research is needed. Therefore, maintaining insecticidal activity while avoiding the production of toxins and allergens of A. flavus will be the future research direction. However, the major impediment to fungal penetration is the host’s cuticle which must be broken down to ease penetration. Fungal pathogens secrete a certain class of cuticle degrading enzymes (protease, chitinase, and lipase) to help dissolve the host integument, thereby overcoming the barrier [ 40 ]. In a previous study, El-Sayed et al. [ 41 ] established that successful penetration and infection of the host cuticle depends on the combined outcome of enzymatic degradation and mechanical pressure exerted by the infecting fungus. Yet, critical questions persist regarding the compatibility of A. flavus pathogenic mechanisms on insects and its effects on humans. A crucial frontier of inquiry beckons: does the application of A. flavus as a biological insecticide in agricultural settings pose any risks of infection to humans and animals? The pursuit of answers to these pivotal queries stands as an imperative next step in unlocking the full potential of A. flavus as a transformative force in sustainable pest management practices. 5. Conclusion In this study, we identified an insect pathogenic fungus A. flavus that we investigated for potential use in detrimentally affecting different developmental stages of D. indica and explored the effect of spore concentration on insect mortality, showing concentration-dependent mortality rates of D. indica larvae and adults, reaching up to 86.65% at the highest concentration. External symptoms observed in infected larvae and adults, including reduced mobility and darkened pigmentation, along with inhibited hatching and green hyphae emergence in eggs, underscored the efficacy of A. flavus 'PP125556' across various developmental stages. Our study thus provides a potential biocontrol insecticide for controlling harmful insects. Declarations Conflict of Interest The authors declare that they have no conflict of interest. Funding: This research was funded by grants from the Jiangsu provincial Independent Innovation Fund (CX༈22)3011༉ References Pal A et al (2020) Cultivation of cucumber in greenhouse. Protected cultivation and smart agriculture, vol 10. New Delhi, New Delhi, India. doi,, pp 139–145 Mallick PK (2022) Evaluating potential importance of cucumber (Cucumis sativus L.-Cucurbitaceae): a brief review. Int J Appl Sci Biotechnol 10(1):12–15 Everatt M, Korycinska A, Malumphy C (2015) Cucurbit moths: Diaphania species. Diaphania species, Cucurbit moths Roopa H et al (2014) Screening on biopesticides of Diaphania indica (Saunders)(Lepidoptera: Pyralidae), a pest of gherkins. Trends Life Sci (India) 29:37–43 Soumya K et al (2017) Integrated Pest Management of melon borer, Diaphania indica (Lepidoptera: Pyralidae) in bittergourd. J Biol Control, : p. 240–244 Umamaheswari R et al (2020) Biotic Stress Management in Horticultural Crops Using Microbial Intervention. Soil and Plant Functions, Rhizosphere Microbes, pp 619–654 Feng M, Poprawski T, Khachatourians GG (1994) Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: current status. Biocontrol Sci Technol 4(1):3–34 Kaur M et al (2021) Aspergillus flavus induced oxidative stress and immunosuppressive activity in Spodoptera litura as well as safety for mammals. BMC Microbiol 21(1):180 Mamo FT et al Study on distribution and characterization of Aspergillus flavus strains to screen potential biocontrol agents from different geographical locations of China. 中国菌物学会 2016 年学术年会论文摘要集, 2016. Naz I et al (2021) Biological control of root knot nematode, Meloidogyne incognita, in vitro, greenhouse and field in cucumber. Biol Control 152:104429 Seye F et al (2014) Effect of entomopathogenic Aspergillus strains against the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae). Appl Entomol Zool 49:453–458 Chen B, Li ZY, Feng MG (2008) Occurrence of entomopathogenic fungi in migratory alate aphids in Yunnan Province of China. Biocontrol 53:317–326 Besas JR, Dizon EI (2014) Biochemical changes of salt-fermented tuna viscera (Dayok) and its effect on histamine content during fermentation. in Int Conf Food Secur Nutr Nafisa G, Hidana R, Virgianti DP (2019) Influence of the Growth of Candida albicans on Several Alternative Medium . in 2nd Bakti Tunas Husada-Health Science International Conference (BTH-HSIC 2020. Atlantis Press de Aldana BRV et al (2021) Screening fungal endophytes from a wild grass for growth promotion in tritordeum, an agricultural cereal. Plant Sci 303:110762 Samson RA et al (2014) Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol 78(1):141–173 White TJ et al (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18(1): pp. 315–322 Sun X et al (2016) Screening of tropical isolates of Metarhizium anisopliae for virulence to the red palm weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae). SpringerPlus 5(1):1–5 Miranda-Miranda E et al (2012) Natural occurrence of lethal aspergillosis in the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Parasitology 139(2):259–263 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425 Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution, 33(7): pp. 1870–1874 Ganehiarachchi G (1997) Aspects of the biology of Diaphania indica (Lepidoptera: Pyralidae). Abbott WS (1925) A method of computing the effectiveness of an insecticide. J econ Entomol 18(2):265–267 Bhuyan KK et al (2017) Evaluation of indigenous biopesticides against Red Spider Mite, Oligonychus coffeae (Nietner) in tea. Mortality 10:100 Martínez LC et al (2022) Susceptibility of Demotispa neivai (Coleoptera: Chrysomelidae) to Beauveria bassiana and Metarhizium anisopliae entomopathogenic fungal isolates. Pest Manag Sci 78(1):126–133 Karthi S et al (2018) Effect of Aspergillus flavus on the mortality and activity of antioxidant enzymes of Spodoptera litura Fab.(Lepidoptera: Noctuidae) larvae. Pestic Biochem Physiol 149:54–60 Zimmermann G (2007) Review on safety of the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Sci Technol 17(9):879–920 Bamisile BS et al (2021) Model application of entomopathogenic fungi as alternatives to chemical pesticides: Prospects, challenges, and insights for next-generation sustainable agriculture. Front Plant Sci 12:741804 Lin W-J et al (2021) Efficacy of Entomopathogenic fungus Aspergillus nomius against Dolichoderus thoracicus. Biocontrol 66:463–473 Yan J et al (2022) First Record of Aspergillus fijiensis as an Entomopathogenic Fungus against Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). J Fungi 8(11):1222 Zhang P et al (2015) First record of Aspergillus oryzae (Eurotiales: Trichocomaceae) as an entomopathogenic fungus of the locust, Locusta migratoria (Orthoptera: Acrididae). Biocontrol Sci Technol 25(11):1285–1298 Khun KK et al (2021) Transmission of Metarhizium anisopliae and Beauveria bassiana to adults of Kuschelorhynchus macadamiae (Coleoptera: Curculionidae) from infected adults and conidiated cadavers. Sci Rep 11(1):2188 Balogun S, Fagade O (2004) Entomopathogenic fungi in population of Zonocerus variegatus (l) in Ibadan, south west, Nigeria. Afr J Biotechnol 3(8):382–386 Scully LR, Bidochka MJ (2005) Serial passage of the opportunistic pathogen Aspergillus flavus through an insect host yields decreased saprobic capacity. Can J Microbiol 51(2):185–189 St. Leger RJ, Screen SE, Shams-Pirzadeh B (2000) Lack of host specialization in Aspergillus flavus. Appl Environ Microbiol 66(1):320–324 Klich MA (2002) Identification of common Aspergillus species. CBS Wang S et al (2022) Glutamine synthetase contributes to the regulation of growth, conidiation, sclerotia development, and resistance to oxidative stress in the fungus Aspergillus flavus. Toxins 14(12):822 Karthaus M (2010) Leitliniengerechte Therapie der invasiven Aspergillose. Mycoses 53:36–43 Kim YH et al (2022) A time-dependently regulated gene network reveals that Aspergillus protease affects mitochondrial metabolism and airway epithelial cell barrier function via mitochondrial oxidants. Free Radic Biol Med 185:76–89 de Krieger C, Schrank A, Vainstein MH (2003) Regulation of extracellular chitinases and proteases in the entomopathogen and acaricide Metarhizium anisopliae. Curr Microbiol 46:0205–0210 El-Sayed G et al (1989) Chitinolytic activity and virulence associated with native and mutant isolates of an entomopathogenic fungus, Nomuraea rileyi. J Invertebr Pathol 54(3):394–403 Supplementary Files Suplementalfile.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4296110","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":306298682,"identity":"64daa638-9096-4cbc-9b15-2cbe08aa047f","order_by":0,"name":"mofeed Abdelhamed Aboelhassan Askar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYFACxgcMDGxAmgeIP4AYDAmEtDAbwLUwziBZCzMPMVp025sZH3woY5CX7zn87LNN2WEGfvYcA4YPv3BrMTtzmNlwxjkGww1n24xn55w7zCDZ88aAcWYfHi038o9J87b9Z9zAz2DMnNt2mMHgRo4BM28PPi3J7L//tjHYz+9n/8xsCdRiD9LyF78WNmbGNobEhrM9xkAG0BYJoBaGH/j9ItlzjiF5w5kzxYw959J5JM48KzjY24BHy/Fmxg8/yhhs5/ekb2b4UWYtx9+evPHBjz+4tWAAUCJgOAB0KsmAFFtGwSgYBaNguAMAuyBRP8acFPAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-7551-5522","institution":"Yangzhou University","correspondingAuthor":true,"prefix":"","firstName":"mofeed","middleName":"Abdelhamed Aboelhassan","lastName":"Askar","suffix":""},{"id":306298683,"identity":"b715bb48-6148-4832-9992-6e0fd16d55b1","order_by":1,"name":"Chen Chen","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Chen","suffix":""},{"id":306298684,"identity":"ef15fd98-1811-4f2f-9463-45d98fadbd3a","order_by":2,"name":"Ali Borham","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Borham","suffix":""},{"id":306298685,"identity":"07646658-3b49-4106-b768-f987d174de51","order_by":3,"name":"Xijun Chen","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xijun","middleName":"","lastName":"Chen","suffix":""},{"id":306298686,"identity":"eb4e649a-4a05-449f-b41e-3fd208204af0","order_by":4,"name":"Huangui Ling","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Huangui","middleName":"","lastName":"Ling","suffix":""},{"id":306298687,"identity":"c108b9dd-2b67-46d5-9440-d1adc2e647aa","order_by":5,"name":"Honghua SU","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Honghua","middleName":"","lastName":"SU","suffix":""}],"badges":[],"createdAt":"2024-04-20 06:25:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4296110/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4296110/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57848798,"identity":"6d21e94d-ddb2-478d-89ef-ff5c60272b93","added_by":"auto","created_at":"2024-06-06 11:23:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":771541,"visible":true,"origin":"","legend":"\u003cp\u003eColony morphology and microscopic examination of \u003cem\u003eA. flavus\u003c/em\u003e ‘PP125556’. Note:\u003c/p\u003e\n\u003cp\u003e(A) ‘PP125556’colony morphology after 7 d of growth; (B) conidial head on MEA (C) cephalic stalk of strain ‘PP125556’ (×400); (D) sporophore of strain ‘PP125556’ (×400); (E) conidia of strain ‘PP125556’ (×400);\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4296110/v1/9828c509ef7b239cf11d9070.png"},{"id":57847948,"identity":"b46d40a4-18d5-4a73-a1b0-e45cefd18c15","added_by":"auto","created_at":"2024-06-06 11:15:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115260,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetictree based on combined sequence analysis of ITS and CaM genes\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4296110/v1/61dffa56ab82a8368f4281bf.png"},{"id":57848796,"identity":"5b155218-bef8-41d4-9eaa-3cb9664f602e","added_by":"auto","created_at":"2024-06-06 11:23:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1. \u003c/strong\u003eVirulence of isolate of \u003cem\u003eA.flavus\u003c/em\u003e against \u003cem\u003eD. indica\u003c/em\u003e larvae treated-with concentrations of 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e spores/mL. Mortality of the pest was observed after every 24 h for 5 days post-treatment.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4296110/v1/8185ebd2de497e35e94b537e.png"},{"id":57848797,"identity":"95da9bfd-dccb-47f1-a2b8-e57c284f57e9","added_by":"auto","created_at":"2024-06-06 11:23:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2. \u003c/strong\u003eVirulence of isolate of \u003cem\u003eA.flavus\u003c/em\u003e against \u003cem\u003eD. indica\u003c/em\u003e adults treated-with concentrations of 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e spores/mL. Mortality of the pest was observed after every 24 h for 3 days post-treatment.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4296110/v1/8ffe008b184a959ea5f82625.png"},{"id":57847951,"identity":"d8da49c6-6926-48e9-bb7a-210091d60084","added_by":"auto","created_at":"2024-06-06 11:15:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1032960,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 3: External symptoms of \u003cem\u003eD. indica\u003c/em\u003elarva with \u003cem\u003eA. flavus\u003c/em\u003e. Note: (A)\u003cem\u003e D. indica\u003c/em\u003e eggs with green mycelium attached. (B) larva infected with strain ‘PP125556’ for 24 h exhibited no change in body color; (C\u003cem\u003e) D. indica\u003c/em\u003einfected with strain ‘PP125556’ for 48 h, with mycelium attached to the \u003cem\u003eD. indica\u003c/em\u003e abdomen resulting in a darker color; (D) \u003cem\u003eD. indica\u003c/em\u003e infected for 48 h exhibited significantly decreased mobility and stiffened limbs; (D) \u003cem\u003eD. indica\u003c/em\u003e infected with strain ‘PP125556’ for 72 h, with mycelium attached to the \u003cem\u003eD. indica\u003c/em\u003e head, abdomen, and thorax, resulting in shrinkage.(E)\u003cem\u003eD. indica\u003c/em\u003e adult with green mycelium attached.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4296110/v1/c5fd52d771196f3b364dd49c.png"},{"id":59361273,"identity":"7d32a892-ccac-4add-83dd-c48cfac1fea3","added_by":"auto","created_at":"2024-06-30 18:28:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3129835,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4296110/v1/27cd75f4-15e6-48ed-89f8-b1064109998f.pdf"},{"id":57847950,"identity":"09b49793-f961-4337-8458-1741a9c5e3f1","added_by":"auto","created_at":"2024-06-06 11:15:14","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10218987,"visible":true,"origin":"","legend":"","description":"","filename":"Suplementalfile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4296110/v1/a81a70ba00fbe087d3ab020a.xlsx"}],"financialInterests":"","formattedTitle":"Aspergillus flavus as an Entomopathogen Infecting Diaphania indica and Control Efficacy Across Different Developmental Stages","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlobally, cucurbitaceous vegetables hold a significant position in both production and consumption, constituting a substantial portion of the vegetable crop market. The Cucurbitaceae family encompasses a diverse range of vegetable crops cultivated for both culinary and medicinal purposes. These crops yield leaves, flowers, fruit, seeds, and roots, each possessing various pharmacological and pharmaceutical properties. Across culinary traditions, cucurbits feature prominently, serving as salad staples like cucumber, gherkin, and long melon, or finding utility in pickled forms such as cucumber, gherkin, and bitter gourd. Additionally, certain cucurbits like muskmelon and watermelon are enjoyed as dessert fruits, while others like ash gourd are transformed into candied or preserved delicacies. Notably, cucumber seeds are valued for their oil, which is recognized for its beneficial effects on brain and body health [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Originating in Asia, cucumbers have a rich history spanning over 3000 years, introduced to China around 100 B.C. and later reaching France by the 9th century [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHosseinzade et al. (2014) corroborated the infestation of Cucurbitaceae by Diaphania species, noting that the larvae target young fruit after consuming the leaves entirely. \u003cem\u003eCucumis sativus L.\u003c/em\u003e, \u003cem\u003eSicyos angulatus L.\u003c/em\u003e, \u003cem\u003eLuffa cylindrica L.\u003c/em\u003e, and various gourds including \u003cem\u003eMomordica charantia L\u003c/em\u003e. and \u003cem\u003eCitrullus lanatus Thunb\u003c/em\u003e are among the preferred hosts for Diaphania species. larvae. Additionally, [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] highlighted the prolific reproductive nature of Diaphania indica populations, particularly in regions such as Hainan Province, China, where multiple generations per year, possibly exceeding ten, are observed. Active D. indica populations persist throughout the year, peaking between April and September. Larval feeding activities not only lead to leaf skeletonization but also pose significant threats to flower and fruit development, often rendering them unmarketable due to secondary pathogen infections. Silk webs formed over entry holes further obstruct natural enemies' access, further exacerbating fruit-feeding damage.\u003c/p\u003e \u003cp\u003eThe importance of entomopathogenic fungi as alternative pest control agents continues to escalate, offering viable solutions to pressing agricultural challenges. Previous research has extensively utilized four bio-pesticides \u003cem\u003eBeauveria bassiana\u003c/em\u003e, \u003cem\u003eNomuraea rileyi, Bacillus thuringiensis\u003c/em\u003e, and Helicoverpa armigera Nucleopolyhedrovirus (HaNPV) against \u003cem\u003eDiaphania indica\u003c/em\u003e, demonstrating their effectiveness and ability to induce high mortality rates [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] underscored the limited success of biological control agents, highlighting the entomopathogenic nematode \u003cem\u003eSteinernema carpocapsae\u003c/em\u003e as a promising candidate. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] evaluated various integrated pest management (IPM) strategies, revealing \u003cem\u003eD. stantoni\u003c/em\u003e, \u003cem\u003eN. rileyi, and B. bassiana\u003c/em\u003e as effective controls compared to alternative approaches. Confirming these findings, [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] emphasized the efficacy of different IPM applications utilizing \u003cem\u003eDolichogenidea stantoni, Trichogramma chilonis, N. rileyi, B. bassiana, Metarhizium anisopliae\u003c/em\u003e, and \u003cem\u003eB. thuringiensis\u003c/em\u003e for managing \u003cem\u003eD. indica\u003c/em\u003e in bitter gourd, providing substantial control over the pest population. These mycopesticides primarily utilize propagules such as conidia, blastospores, and hyphae, offering immediate pest eradication and triggering secondary infection by dispersing mycotic spores horizontally from cadavers. Notably, wettable powder-based biopesticides have harnessed these fungi, further emphasizing their potential in sustainable pest management strategies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAspergillus flavus is an important entomopathogenic fungi [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] observed that A. flavus impacts \u003cem\u003eSpodoptera litura\u003c/em\u003e by directly influencing its immune system, leading to a reduction in immune functionality. A. flavus specifically targets third and fourth instar larvae of \u003cem\u003eS. litura.\u003c/em\u003e According to preliminary findings, the cosmopolitan saprophytic fungus known as \u003cem\u003eA. flavus\u003c/em\u003e shows potential as a promising agent for controlling aphids [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Initial laboratory experiments revealed that \u003cem\u003eA. flavus\u003c/em\u003e can effectively target aphids, with a mortality rate observed at concentrations of 1.23 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e spores/mL (LC50) and 1.34 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e spores/mL (LC90) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, in a separate investigation focusing on cabbage and wheat, significant susceptibility to fungal biocontrol was observed, with 33% of cabbage aphids and 37% of wheat aphids affected, and \u003cem\u003eA. flavus\u003c/em\u003e demonstrat notable efficacy among the pathogens tested [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nonetheless, the precise mechanism through which \u003cem\u003eA. flavus\u003c/em\u003e regulates aphid populations, as well as the specific active components involved, remains largely unexplored.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Isolation and Identification of the Strain\u003c/h2\u003e \u003cp\u003eIn August 2023, we isolated a strain from infected \u003cem\u003eDiaphania indica\u003c/em\u003e eggs collected from Yangzhou, Jiangsu Province, China, and inoculated it on potato dextrose agar medium (PDA) using the spread plate method [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Each gradient was repeated three times and cultured for seven days at 27 ̊C. Purification was repeated until a single colony was obtained [15].\u003c/p\u003e \u003cp\u003eFor the strain, two genes were used for molecular identification according to [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. PCR amplification was performed: (the forward primer and reverse primer were synthesized by Tsingke Biotech Technology, including the internal transcribed spacer (ITS) gene with primers ITS1 (5\u0026prime;-TCCGTAGGTGAACCTGCG-3\u0026prime;) and ITS4 (5\u0026prime;-TCCTCCGC TTATTGATATGC-3\u0026prime;), as well as a segment of the calmodulin gene (CaM) with primers cmd5 (5\u0026prime;-CCGAGTACAAGGAGGCCTTC-3\u0026prime;) and cmd6 (5\u0026prime;-CCGATAG AGGTCATAACGTGG-3\u0026prime;) (submitted separately to GenBank with accession numbers PP125556 for ITS and PP464989 for \u003cem\u003eCaM\u003c/em\u003e). The PCR protocol was conducted as described by White and Bruns [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Sequences were aligned and compared to existing sequence data in the GenBank database using the Basic Local Alignment Search Tool (NCBI BLAST) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Microscopy Identification\u003c/h2\u003e \u003cp\u003eThe mycelium obtained from the Petri dishes was cultured on microscope glass slides covered with a thin layer of Sabouraud agar, following a method previously described by [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The cultures were then incubated at 27 ̊ C for three days. Afterwards, the fungal morphology and the cultures on the microscope glass slides were identified using microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eITS and \u003cem\u003eCaM\u003c/em\u003e sequence data of the isolate were subjected to phylogenetic analysis by comparison with registered sequences in the GenBank database. The ITS and CaM sequences were concatenated by using PhyloSuite software and the tree was built by MEGA using the NJ method [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Before the phylogenetic tree was built using MEGA 7.0 software, which provided the topology and length of the branches, ITS and \u003cem\u003eCaM\u003c/em\u003e sequences were aligned, and superfluous sections were deleted [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Mass Rearing of Test Insects\u003c/h2\u003e \u003cp\u003eThe larvae and pupae of \u003cem\u003eD. indica\u003c/em\u003e were removed from the cucumber plants that were infected to raise the initial culture. They were raised individually in jars (20.0 cm in height and 15.0 cm in diameter), which were regularly supplied with fresh fruit and leaves. The excreta from the half-consumed fruit and leaves were removed. Twenty pairs of newly emerged male and female adults were placed in ten mating jars, each measuring 20.0 cm in height and 15.0 cm in diameter. Fresh cucumber leaves (for laying eggs) and a cotton swab dipped in a 10% diluted honey solution (for feeding the adults) were placed inside these jars. Gravid females produced second-generation (eggs, larvae, and adults), which were used for additional treatments and sprayed with fungus spore solutions. The temperature and relative humidity during the maintenance of culture ranged from 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C to 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 69\u0026thinsp;\u0026plusmn;\u0026thinsp;1 to 70\u0026thinsp;\u0026plusmn;\u0026thinsp;1%, respectively, according to [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Activity of \u003cem\u003eAspergillus flavus\u003c/em\u003e against \u003cem\u003eDiaphania indica\u003c/em\u003e Under Laboratory Conditions\u003c/h2\u003e \u003cp\u003eTo prepare the spore suspension, incubation plates were washed with sterile water on a sterile operating table. The spores were collected in a conical flask with glass beads, shaken thoroughly, and scattered. A single spore suspension was obtained via filtration with a cotton-stuffed syringe. Concentrations were adjusted to concentration to 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e spores/mL. Finally, 0.1% tween-20 was added, and the suspension was refrigerated (4\u0026ordm;C).\u003c/p\u003e \u003cp\u003eFor treatment, three groups of pests were sprayed with solutions containing 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e, and 10\u003csup\u003e9\u003c/sup\u003e spores/mL for each treatment. An additional control group was sprayed without fungal spores for the same timeframe and with the same statistical design as for the treatments. Notably, the control group included four replicas. We did these treatments for eggs, larvae, and adults.For the egg stage, larvae, and adults of \u003cem\u003eD. indica\u003c/em\u003e 60 eggs were counted, 20 larvae \u0026rdquo;3rd instar\u0026rdquo; and 15 healthy adults per four replicates for each concentration were transferred to the treated cucumber leaf. A single dose of 2 ml of suspension of each concentration was sprayed on the surface of the leaf disc and the replicates using a hand spray atomizer at a distance of 25\u0026ndash;30 cm. After the applications, we registered the death rate daily. The mortality percentage of mites was calculated and corrected according to Abbott's formula [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Also, the reduction percentage of hatchability was calculated according to [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical Analyses\u003c/h2\u003e \u003cp\u003eFor statistical analysis, exact numbers were used in order to remove any analytical mistakes. The single-factor analysis of variance was carried out using SPSS 26.0, and the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation was the output. Duncan's technique was applied to several comparisons. The p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 criterion was used to evaluate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Isolation and Identification of the Strain\u003c/h2\u003e \u003cp\u003eFollowing 2\u0026ndash;3 days of cultivation on potato dextrose agar (PDA), the strain manifested loose, flat colonies with regular edges, spanning 2\u0026ndash;3 cm in diameter. Initially, the colonies exhibited a pristine white hue, gradually transitioning to a verdant green at their centers, characterized by a profusion of green spores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,E). Notably, the nutritional hyphae displayed septation, while a segment of the aerial hyphae extended into elongated, rough conidiophores, culminating in nearly spherical apical sacs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, the colony surface bore numerous small peduncles, each adorned with clusters of coarse-surfaced spherical conidia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Conidial heads that were yellow-green when young, shifted to olive-green in age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). DNA fragment sequencing unveiled gene lengths of 559 bp for ITS and 481 bp for CaM in the strain. The DNA sequences were duly submitted to GenBank, acquiring accession numbers (PP125556 for ITS and PP464989 for \u003cem\u003eCaM\u003c/em\u003e). BLASTN comparison against GenBank entries confirmed similarity with the reported \u003cem\u003eA. flavus\u003c/em\u003e strains (Supplemental information). Phylogenetic analysis positioned the strain close to the \u003cem\u003eA. flavus\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), corroborating our morphological identification of the strain as \u003cem\u003eA. flavus\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Biocontrol Efficacy of \u003cem\u003eA. flavus\u003c/em\u003e \u0026lsquo;PP125556\u0026rsquo; and Dose\u0026ndash;Response Bioassay\u003c/h2\u003e \u003cp\u003eThe outcomes reveal the mortality percentages of \u003cem\u003eD. indica\u003c/em\u003e subsequent to the application of varying concentrations of conidia/ml, as depicted in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Significantly, the results unveiled a marked influence on mortality rates relative to the concentration of conidia/ml. Notably, T1 (1 x 10⁹ conidia/ml) exhibited the highest mortality rates, recording 53.06% \u0026plusmn; 4.71 for eggs, 70.57% \u0026plusmn; 15.81 for larvae, and 86.65% \u0026plusmn; 6.42 for adults. These values were notably distinct from those of the other treatments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, T2 (1 x 10⁸ conidia/ml) and T3 (1 x 10⁷ conidia/ml) demonstrated considerable mortality rates, with 41.22% \u0026plusmn; 8.67 and 30.20% \u0026plusmn; 4.32 for eggs, 56.22% \u0026plusmn; 16.29 and 39.47% \u0026plusmn; 4.58 for larvae, and 69.00% \u0026plusmn; 3.21 and 41.72% \u0026plusmn; 3.71 for adults, respectively. Conversely, the control treatment (T4) yielded the lowest mortality rates, registering 1.25% \u0026plusmn; 1.59 for eggs, 3.75% \u0026plusmn; 2.50 for larvae, and 3.34% \u0026plusmn; 3.85 for adults. These values exhibited significant deviations from those of the treated groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Thus, the findings underscore a concentration-dependent effect on \u003cem\u003eD. indica\u003c/em\u003e mortality rates, with higher concentrations correlating to increased mortality.\u003c/p\u003e \u003cp\u003eThe study underscores the importance of dosage precision in entomopathogenic fungal applications for effective pest management. Notably, treatments with higher concentrations of conidia/ml exhibited pronounced mortality effects on \u003cem\u003eD. indica\u003c/em\u003e across all developmental stages, indicative of the potential for targeted control strategies. These findings contribute valuable insights into optimizing fungal biopesticide formulations and deployment protocols for enhanced efficacy in integrated pest management programs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of different concentrations of \u003cem\u003eAspergillus flavus\u003c/em\u003e on \u003cem\u003eDiaphania indica\u003c/em\u003e mortality\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConc. (conidia/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMortality % of \u003cem\u003eDiaphania indica\u003c/em\u003e After Applications\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEgg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLarvae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdult\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eT1(1 x 10 \u003csup\u003e9\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.06\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;15.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;6.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eT2(1 x 10 \u003csup\u003e8\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.22\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;8.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;16.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;3.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eT3(1 x 10 \u003csup\u003e7\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.20\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.47\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.72\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;3.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eT4 (Control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.75\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.34\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;3.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are means of replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u0026minus;h\u003c/sup\u003e means within a column with different letters are significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003cp\u003eThese findings highlight the intricate interplay between temporal dynamics and treatment modalities in shaping mortality patterns within the targeted pest populations, thus providing valuable insights for optimizing pest management strategies Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrate the temporal progression of mortality rates for each treatment (T1, T2, T3, and T4) administered to the specified larval and adult stages, respectively. Within Treatment T1, there is a discernible and consistent escalation in mortality percentage observed throughout the duration of the study, indicative of a progressive impact on the targeted larval and adult populations. Similarly, Treatment T2 manifests an ascending trend in mortality rates over the observation period, albeit potentially at a different pace compared to T1, reflecting the temporal dependency of treatment efficacy. Treatment T3 also showcases an increase in mortality percentages over time, although the rate of ascent may be relatively subdued in comparison to T1 and T2. In contrast, Treatment T4, functioning as the control, exhibits relatively static or minimal mortality percentages across the monitored duration, signalling negligible effects on pest mortality over time.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. External Symptoms of \u003cem\u003eD. indica\u003c/em\u003e Infected with \u003cem\u003eA. flavus\u003c/em\u003e \u0026lsquo;PP125556\u0026rsquo;\u003c/h2\u003e \u003cp\u003eExamination of eggs under a stereomicroscope three days post-infection revealed no hatching, while after two days, green hyphae were observed emerging from the eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, Examination under a stereomicroscope unveiled negligible changes in the mobtility or physical attributes of \u003cem\u003eD. indica\u003c/em\u003e larvae 24 hours post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Nonetheless, a notable reduction in \u003cem\u003eD. indica\u003c/em\u003e mobility was discernible after 48 hours of infection, concomitant with darkened body pigmentation and the emergence of black intersegmental folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Following 72 hours of infection, white hyphae proliferated on the head, abdomen, and thorax of \u003cem\u003eD. indica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), indicating their demise [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, their bodies displayed signs of desiccation and stiffness. Upon inspecting adults, no significant alterations in mobility were noted after 24 hours, but after 48 hours we started to record mortalities; however, when placed in moist cotton, evidence of infection surfaced, characterized by white hyphae colonization on their head, abdomen, and thorax, subsequently transitioning to green (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study delved into the isolation of \u003cem\u003eA. flavus\u003c/em\u003e from \u003cem\u003eD. indica\u003c/em\u003e eggs, aiming to understand its role in biocontrol efficacy. By meticulously characterizing \u003cem\u003eA. flavus\u003c/em\u003e from these eggs, we sought to elucidate its potential in combatting \u003cem\u003eD. indica\u003c/em\u003e infestations. Through comprehensive analyses, we aim to uncover the mechanisms underlying \u003cem\u003eA. flavus\u003c/em\u003e interaction with \u003cem\u003eD. indica\u003c/em\u003e populations, contributing to the development of sustainable strategies for pest management. The insights gained from this research hold promise for the formulation of targeted and environmentally friendly approaches to controlling \u003cem\u003eD. indica\u003c/em\u003e, benefiting agricultural and ecological systems while advancing our understanding of fungal biocontrol agents.\u003c/p\u003e \u003cp\u003eEntomopathogenic fungi (EPF) strains serve as indispensable assets in combatting insect pests, handling yield losses, and averting quality deterioration in agricultural and forestry domains. These fungi, heralded as valuable alternatives to traditional synthetic insecticides, are widely deployed to efficiently manage pests within agroecosystems [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]Notably, compared to other microbial adversaries, EPFs offer multifaceted advantages in pest control applications [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Despite insects' intricate arrays of defense mechanisms against infective agents, pathogens have undergone coevolution to circumvent these defenses, emphasizing the dynamic interplay in host-pathogen interactions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eWhile research predominantly focuses on EPF resources like \u003cem\u003eBeauveria species, Metarhizium species\u003c/em\u003e, and \u003cem\u003eLecanicillium species.\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], further exploration of diverse EPFs is imperative for bolstering pest management efficacy. Enter the diverse realm of Aspergillus species, renowned for their biological versatility and multifaceted utility. Notably, certain Aspergillus species, including \u003cem\u003eA. flavus, A. nomius, A. fijiensis\u003c/em\u003e, and \u003cem\u003eA. oryzae\u003c/em\u003e, have demonstrated remarkable efficacy against a spectrum of insect pests including Locusta migratoria, \u003cem\u003eSpodoptera litura, Diaphorina citri\u003c/em\u003e, and \u003cem\u003eDolichoderus thoracicus\u003c/em\u003e [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our study showed the revelation of the isolation and identification of a potent \u003cem\u003eA. flavus\u003c/em\u003e strain from infected \u003cem\u003eD. indica\u003c/em\u003e eggs, wielding formidable pathogenicity against agricultural insect pests. The \u003cem\u003eA. flavus\u003c/em\u003e strain emerges as a promising candidate for broader spectrum EPF deployment in \u003cem\u003eD. indica\u003c/em\u003e control strategies. \u003cem\u003eD. indica\u003c/em\u003e infected via spore dissemination may act as reservoirs for perpetuating infection and facilitating further incursions into additional \u003cem\u003eD. indica\u003c/em\u003e populations [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]The present study confirms that the fungal isolate of \u003cem\u003eA. flavus\u003c/em\u003e is a potential biocontrol agent for the management of \u003cem\u003eD. indica\u003c/em\u003e. The mortalities of these pests were found to be proportional to the conidial concentrations used in the bioassay involving the fungal isolate.\u003c/p\u003e \u003cp\u003eThe efficacy of \u003cem\u003eA. flavus\u003c/em\u003e was previously studied against mango seed weevil, \u003cem\u003eSternochetus mangiferae (Coleoptera: Curculionidae)\u003c/em\u003e, producing mortality rates of 80% at a conidial concentration of 6.8\u0026times;10\u003csup\u003e7\u003c/sup\u003e conidia/mL. Aspergillus species have been reported to kill the tropical locust Zonocerus variegatus and cause infection among many insect populations [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, these Aspergillus species are unknown whether it is host-specific. Previous studies have reported the effectiveness of \u003cem\u003eA. flavus\u003c/em\u003e against a wide range of insects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. \u003cem\u003eA. flavus\u003c/em\u003e was also tested against \u003cem\u003eGalleria mellonella\u003c/em\u003e, killing 100% of insects after 48 h when injected with 3 \u0026times; 103 conidia/mL of the fungus. In contrast, \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eA. nidulans\u003c/em\u003e lacked parasitic attributes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, entomopathogenic fungi have gained attention as potential biocontrol agents, yet their safety and mode of action remain unclear. For application of \u003cem\u003eA. flavus\u003c/em\u003e to pest management, two problems should be considered. \u003cem\u003eA. flavus\u003c/em\u003e could produce a variety of mycotoxins, such as aflatoxins, cyclopiazonic acid and 3-nitropropionic acid, that have strong carcinogenic, teratogenic, and organ necrosis properties that threaten the food safety of agricultural products [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. On the other hand, this kind of fungus may cause human invasive aspergillosis or allergenic problems [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] explored the toxicity of \u003cem\u003eA. flavus\u003c/em\u003e on \u003cem\u003eS. litura\u003c/em\u003e, analyzing its effects on antioxidant and immune defenses. It showed increased antioxidant activity and negative impacts on haemocytes. However, genotoxicity assessments in rats showed no significant effects, suggesting further research is needed. Therefore, maintaining insecticidal activity while avoiding the production of toxins and allergens of \u003cem\u003eA. flavus\u003c/em\u003e will be the future research direction.\u003c/p\u003e \u003cp\u003eHowever, the major impediment to fungal penetration is the host\u0026rsquo;s cuticle which must be broken down to ease penetration. Fungal pathogens secrete a certain class of cuticle degrading enzymes (protease, chitinase, and lipase) to help dissolve the host integument, thereby overcoming the barrier [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In a previous study, El-Sayed et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] established that successful penetration and infection of the host cuticle depends on the combined outcome of enzymatic degradation and mechanical pressure exerted by the infecting fungus. Yet, critical questions persist regarding the compatibility of \u003cem\u003eA. flavus\u003c/em\u003e pathogenic mechanisms on insects and its effects on humans. A crucial frontier of inquiry beckons: does the application of \u003cem\u003eA. flavus\u003c/em\u003e as a biological insecticide in agricultural settings pose any risks of infection to humans and animals? The pursuit of answers to these pivotal queries stands as an imperative next step in unlocking the full potential of \u003cem\u003eA. flavus\u003c/em\u003e as a transformative force in sustainable pest management practices.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, we identified an insect pathogenic fungus A. flavus that we investigated for potential use in detrimentally affecting different developmental stages of \u003cem\u003eD. indica\u003c/em\u003e and explored the effect of spore concentration on insect mortality, showing concentration-dependent mortality rates of D. indica larvae and adults, reaching up to 86.65% at the highest concentration. External symptoms observed in infected larvae and adults, including reduced mobility and darkened pigmentation, along with inhibited hatching and green hyphae emergence in eggs, underscored the efficacy of A. flavus 'PP125556' across various developmental stages. Our study thus provides a potential biocontrol insecticide for controlling harmful insects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by grants from the Jiangsu provincial Independent Innovation Fund (CX༈22)3011༉\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePal A et al (2020) \u003cem\u003eCultivation of cucumber in greenhouse.\u003c/em\u003e Protected cultivation and smart agriculture, vol 10. New Delhi, New Delhi, India. doi,, pp 139\u0026ndash;145\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMallick PK (2022) Evaluating potential importance of cucumber (Cucumis sativus L.-Cucurbitaceae): a brief review. Int J Appl Sci Biotechnol 10(1):12\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEveratt M, Korycinska A, Malumphy C (2015) Cucurbit moths: Diaphania species. Diaphania species, Cucurbit moths\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoopa H et al (2014) Screening on biopesticides of Diaphania indica (Saunders)(Lepidoptera: Pyralidae), a pest of gherkins. Trends Life Sci (India) 29:37\u0026ndash;43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoumya K et al (2017) Integrated Pest Management of melon borer, Diaphania indica (Lepidoptera: Pyralidae) in bittergourd. J Biol Control, : p. 240\u0026ndash;244\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmamaheswari R et al (2020) Biotic Stress Management in Horticultural Crops Using Microbial Intervention. Soil and Plant Functions, Rhizosphere Microbes, pp 619\u0026ndash;654\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng M, Poprawski T, Khachatourians GG (1994) Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: current status. Biocontrol Sci Technol 4(1):3\u0026ndash;34\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaur M et al (2021) Aspergillus flavus induced oxidative stress and immunosuppressive activity in Spodoptera litura as well as safety for mammals. BMC Microbiol 21(1):180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMamo FT et al Study on distribution and characterization of Aspergillus flavus strains to screen potential biocontrol agents from different geographical locations of China. 中国菌物学会 2016 年学术年会论文摘要集, 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaz I et al (2021) Biological control of root knot nematode, Meloidogyne incognita, in vitro, greenhouse and field in cucumber. Biol Control 152:104429\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeye F et al (2014) Effect of entomopathogenic Aspergillus strains against the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae). Appl Entomol Zool 49:453\u0026ndash;458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen B, Li ZY, Feng MG (2008) Occurrence of entomopathogenic fungi in migratory alate aphids in Yunnan Province of China. Biocontrol 53:317\u0026ndash;326\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBesas JR, Dizon EI (2014) Biochemical changes of salt-fermented tuna viscera (Dayok) and its effect on histamine content during fermentation. in Int Conf Food Secur Nutr\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNafisa G, Hidana R, Virgianti DP (2019) \u003cem\u003eInfluence of the Growth of Candida albicans on Several Alternative Medium\u003c/em\u003e. in \u003cem\u003e2nd Bakti Tunas Husada-Health Science International Conference (BTH-HSIC\u003c/em\u003e 2020. Atlantis Press\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Aldana BRV et al (2021) Screening fungal endophytes from a wild grass for growth promotion in tritordeum, an agricultural cereal. Plant Sci 303:110762\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamson RA et al (2014) Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol 78(1):141\u0026ndash;173\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite TJ et al (1990) \u003cem\u003eAmplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.\u003c/em\u003e PCR protocols: a guide to methods and applications, 18(1): pp. 315\u0026ndash;322\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun X et al (2016) Screening of tropical isolates of Metarhizium anisopliae for virulence to the red palm weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae). SpringerPlus 5(1):1\u0026ndash;5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiranda-Miranda E et al (2012) Natural occurrence of lethal aspergillosis in the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Parasitology 139(2):259\u0026ndash;263\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406\u0026ndash;425\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Tamura K (2016) \u003cem\u003eMEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets.\u003c/em\u003e Molecular biology and evolution, 33(7): pp. 1870\u0026ndash;1874\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanehiarachchi G (1997) \u003cem\u003eAspects of the biology of Diaphania indica (Lepidoptera: Pyralidae).\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbbott WS (1925) A method of computing the effectiveness of an insecticide. J econ Entomol 18(2):265\u0026ndash;267\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhuyan KK et al (2017) Evaluation of indigenous biopesticides against Red Spider Mite, Oligonychus coffeae (Nietner) in tea. Mortality 10:100\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez LC et al (2022) Susceptibility of Demotispa neivai (Coleoptera: Chrysomelidae) to Beauveria bassiana and Metarhizium anisopliae entomopathogenic fungal isolates. Pest Manag Sci 78(1):126\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarthi S et al (2018) Effect of Aspergillus flavus on the mortality and activity of antioxidant enzymes of Spodoptera litura Fab.(Lepidoptera: Noctuidae) larvae. Pestic Biochem Physiol 149:54\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmermann G (2007) Review on safety of the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Sci Technol 17(9):879\u0026ndash;920\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBamisile BS et al (2021) Model application of entomopathogenic fungi as alternatives to chemical pesticides: Prospects, challenges, and insights for next-generation sustainable agriculture. Front Plant Sci 12:741804\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin W-J et al (2021) Efficacy of Entomopathogenic fungus Aspergillus nomius against Dolichoderus thoracicus. Biocontrol 66:463\u0026ndash;473\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan J et al (2022) First Record of Aspergillus fijiensis as an Entomopathogenic Fungus against Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). J Fungi 8(11):1222\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang P et al (2015) First record of Aspergillus oryzae (Eurotiales: Trichocomaceae) as an entomopathogenic fungus of the locust, Locusta migratoria (Orthoptera: Acrididae). Biocontrol Sci Technol 25(11):1285\u0026ndash;1298\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhun KK et al (2021) Transmission of Metarhizium anisopliae and Beauveria bassiana to adults of Kuschelorhynchus macadamiae (Coleoptera: Curculionidae) from infected adults and conidiated cadavers. Sci Rep 11(1):2188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalogun S, Fagade O (2004) Entomopathogenic fungi in population of Zonocerus variegatus (l) in Ibadan, south west, Nigeria. Afr J Biotechnol 3(8):382\u0026ndash;386\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScully LR, Bidochka MJ (2005) Serial passage of the opportunistic pathogen Aspergillus flavus through an insect host yields decreased saprobic capacity. Can J Microbiol 51(2):185\u0026ndash;189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSt. Leger RJ, Screen SE, Shams-Pirzadeh B (2000) Lack of host specialization in Aspergillus flavus. Appl Environ Microbiol 66(1):320\u0026ndash;324\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlich MA (2002) Identification of common Aspergillus species. CBS\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S et al (2022) Glutamine synthetase contributes to the regulation of growth, conidiation, sclerotia development, and resistance to oxidative stress in the fungus Aspergillus flavus. Toxins 14(12):822\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarthaus M (2010) Leitliniengerechte Therapie der invasiven Aspergillose. Mycoses 53:36\u0026ndash;43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim YH et al (2022) A time-dependently regulated gene network reveals that Aspergillus protease affects mitochondrial metabolism and airway epithelial cell barrier function via mitochondrial oxidants. Free Radic Biol Med 185:76\u0026ndash;89\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Krieger C, Schrank A, Vainstein MH (2003) Regulation of extracellular chitinases and proteases in the entomopathogen and acaricide Metarhizium anisopliae. Curr Microbiol 46:0205\u0026ndash;0210\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Sayed G et al (1989) Chitinolytic activity and virulence associated with native and mutant isolates of an entomopathogenic fungus, Nomuraea rileyi. J Invertebr Pathol 54(3):394\u0026ndash;403\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biological control, Diaphania indica, Aspergillus flavus, molecular identification, fungal PCR","lastPublishedDoi":"10.21203/rs.3.rs-4296110/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4296110/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eDiaphania indica\u003c/em\u003e (Lepidoptera: Crambidae) is one of the most important pests infesting many cucurbitaceous vegetables. During the rearing of insect eggs, we observed a fungal infection of these insect eggs. The fungus produces aflatoxins which are considered secondary polyketide metabolites, which cause the death of pests. Therefore, this work aimed to isolate and identify this fungus by amplifying the internal transcribed spacer (ITS) region of the rDNA, as well as evaluating the efficiency of this fungus in control. Aspergillus flavus, 'PP125556,' showcased robust pathogenicity against a range of \u003cem\u003eD. indica\u003c/em\u003e pests. The results showed that colonies of 'PP125556' cultivated on potato dextrose agar (PDA) exhibited distinctive morphological characteristics, transitioning from pristine white to verdant green. Bioassays demonstrated concentration-dependent mortality rates of \u003cem\u003eD. indica\u003c/em\u003e larvae and adults when exposed to varying concentrations of 'PP125556' conidia, with the highest concentration (1x10\u003csup\u003e9\u003c/sup\u003e conidia/ml) inducing significant death with the highest mortality (53.06% for eggs, 70.57% for larvae, and 86.65% for adults). Furthermore, examination under a stereomicroscope revealed conspicuous external symptoms in infected larvae, including reduced mobility, darkened body pigmentation, and the emergence of white hyphae, indicative of mortality. Additionally, infected eggs exhibited inhibited hatching and the emergence of green hyphae, while infected adults displayed mortality and white hyphae colonization, underscoring the potent biocontrol efficacy of \u003cem\u003eA. flavus\u003c/em\u003e 'PP125556' against \u003cem\u003eD. indica\u003c/em\u003e across diverse developmental stages.\u003c/p\u003e","manuscriptTitle":"Aspergillus flavus as an Entomopathogen Infecting Diaphania indica and Control Efficacy Across Different Developmental Stages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-06 11:15:09","doi":"10.21203/rs.3.rs-4296110/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"73f1e03f-e8a7-4962-8c9d-c5ec33f07acb","owner":[],"postedDate":"June 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-30T18:20:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-06 11:15:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4296110","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4296110","identity":"rs-4296110","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}