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

doi:10.21203/rs.3.rs-4296110/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-4296110/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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 (1x10⁹ 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

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 × 10³ spores/mL (LC50) and 1.34 × 10⁷ 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.

2.1. 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 10⁷, 10⁸, and 10⁹ 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 10⁷, 10⁸, and 10⁹ 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.

3.1. 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. 1A,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. 1C). Moreover, the colony surface bore numerous small peduncles, each adorned with clusters of coarse-surfaced spherical conidia (Fig. 1D). Conidial heads that were yellow-green when young, shifted to olive-green in age (Fig. 1B). 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
Conc. (conidia/ml)	Egg	Larvae	Adult
T1(1 x 10 ⁹)	53.06^cd	70.57^b	86.65^a
T1(1 x 10 ⁹)	± 4.71	± 15.81	± 6.42
T2(1 x 10 ⁸)	41.22^de	56.22^c	69.00^b
T2(1 x 10 ⁸)	± 8.67	± 16.29	± 3.21
T3(1 x 10 ⁷)	30.20^e	39.47^e	41.72^de
T3(1 x 10 ⁷)	± 4.32	± 4.58	± 3.71
T4 (Control)	1.25^f	3.75^f	3.34^f
T4 (Control)	± 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. 3A). Furthermore, Examination under a stereomicroscope unveiled negligible changes in the mobtility or physical attributes of D. indica larvae 24 hours post-infection (Fig. 3B). 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. 3C). Following 72 hours of infection, white hyphae proliferated on the head, abdomen, and thorax of D. indica (Fig. 3D), 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. 3E).

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 6.8×10⁷ 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.

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.

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༉

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

Suplementalfile.xlsx

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Isolation and Identification of the Strain

2.2. Microscopy Identification

2.3. Phylogenetic Analysis

2.4. Mass Rearing of Test Insects

2.5. Activity of Aspergillus flavus against Diaphania indica Under Laboratory Conditions

2.6. Statistical Analyses

3. Results

3.1. Isolation and Identification of the Strain

3.2. Biocontrol Efficacy of A. flavus ‘PP125556’ and Dose–Response Bioassay

3.3. External Symptoms of D. indica Infected with A. flavus ‘PP125556’

4. Discussion

5. Conclusion

Declarations

Conflict of Interest

Funding:

References

Supplementary Files

Status:

Version 1