First Record of Clonostachys rosea (Ascomycota: Hypocreales) Entomopathogenic Fungus in the Mango Hopper Amritodus atkinsoni (Hemiptera: Cicadellidae)

Mango hopper (Amritodus atkinsoni Lethierry) causes devastations in the early vegetative stage of the mango crop. The classical management of mango hopper is with systemic insecticides but their overuse has caused environmental pollution. Here, we have evaluated the entomopathogenic role of Clonostachys rosea through bioassay and optimized media for its large-scale culturing. The current study reveals the potentiality of C. rosea as entomopathogenic on A. atkinsoni. Initially, morphological and molecular characterization was used to validate local isolates’ identity as C. rosea. Further, we have evaluated the entomopathogenic role of C. rosea through a bioassay, where the highest mean mortality in A. atkinsoni was observed at a treatment concentration of 3 × 108 conidia/mL, with 96.67% mortality after 168 h of infection. This work also provides insight into the laboratory-based media standardization for C. rosea, resulting in oatmeal agar media and broth as the most suitable artificial media, and 20 °C temperature for its mass culture. Thus, C. rosea is a novo-entomopathogenic fungus on A. atkinsoni and has a high potency to be included in the management of mango hopper pests.


iz University
21589JeddahSaudi Arabia

King Fahd Medical Research Center
Special Infectious Agents Unit
King Abdulaziz University
21589JeddahSaudi Arabia

Yasser S Mostafa 0000-0001-9603-5328
Department of Biology
College of Science
King Khalid University
61413AbhaSaudi Arabia

Sulaiman A Alrumman 0000-0001-6685-5456
Department of Biology
College of Science
King Khalid University
61413AbhaSaudi Arabia

Mahmoud Helal 
Department of Mechanical Engineering
Faculty of Engineering
Taif University
P.O. Box 1109921944TaifSaudi Arabia

Production Engineering and Mechanical Design Department
Faculty of Engineering
Mansoura University
P.O. Box 35516MansouraEgypt


Isabella Pentimone and Lawrence S. Young


First Record of Clonostachys rosea (Ascomycota: Hypocreales) Entomopathogenic Fungus in the Mango Hopper Amritodus atkinsoni (Hemiptera: Cicadellidae)
30 November 2022880015B76BD6C204D4F7EC1468D389E410.3390/pathogens11121447Received: 22 October 2022 Accepted: 28 November 2022Tamta, A.K.Pandey, R.Sharma, J.R.Rai, R.Barman, M.M. G., D.Mitra, D.Mohapatra, P.K.D.Sami, R.Al-Mushhin, A.A.M.et al. First Record of Clonostachys rosea biological controlnovo-entomopathogenic fungusClonostaychs roseamango hoppermedia optimizationentomologyetiology
Mango hopper (Amritodus atkinsoni Lethierry) causes devastations in the early vegetative stage of the mango crop.The classical management of mango hopper is with systemic insecticides but their overuse has caused environmental pollution.Here, we have evaluated the entomopathogenic role of Clonostachys rosea through bioassay and optimized media for its large-scale culturing.The current study reveals the potentiality of C. rosea as entomopathogenic on A. atkinsoni.Initially, morphological and molecular characterization was used to validate local isolates' identity as C. rosea.Further, we have evaluated the entomopathogenic role of C. rosea through a bioassay, where the highest mean mortality in A. atkinsoni was observed at a treatment concentration of 3 × 10 8 conidia/mL, with 96.67% mortality after 168 h of infection.This work also provides insight into the laboratorybased media standardization for C. rosea, resulting in oatmeal agar media and broth as the most suitable ar ificial media, and 20 • C temperature for its mass culture.Thus, C. rosea is a novoentomopathogenic fungus on A. atkinsoni and has a high potency to be included in the management of mango hopper pests.

Introduction

Mangifera indica L. (Sapindales: Anacardiaceae) is a fruit crop that is cultivated in India, China, Thailand, Mexico, Pakistan, the Philippines, Indonesia, Brazil, Nigeria, and Egypt.India is the hub of mango diversity and is also one of the largest producers with an annual production of 18.0 million tons.Though India has the potential for higher production, insect pests act as a stumbling block to obtaining better growth and production of this crop.To date, 400 species of insect pests have been recorded to damage mango [1].Among them, Amritodus atkinsoni Lethierry (Hemiptera: Cicadellidae) is serious insect pests of this crop [2].Active nymphal and adult stages can affect the vigor of the trees by sucking the sap [3][4][5].Several synthetic insecticides have been used to manage such a notorious insect pest [6].Howeve

e of insecticides resulted in deleterious comp
ications such as insect resistance, resurgence, and accumulation of soil and fruit residues [7].These problems can thus be sustainably addressed by developing alternative methods such as biological control agents.In this perspective, screening of the potential entomopathogenic fungus Clonostachys rosea was carried out in this study.This fungus is present in various cultivated and non-cultivated areas, mostly in neutral to alkaline pH soils [8][9][10].This myco-etiological agent has been classified as pathogenic to fungi, as an endophytic-antagonistic in plants, and as entomopathogenic [9][10][11].However, only a few studies have characterized the entomopathogenic nature of this fungus.To date, C. rosea infection has been recorded on Oncometopia tucumana Schröder (Hemiptera: Cicadellidae), Sonesimia grossa Signoret (Hemiptera: Cicadellidae), Anopheles stephensi Liston (Diptera: Culicidae), Culex quinquefasciatus Say (Diptera: Culicidae), Hypera postica Gyllenhal (Coleoptera: Curculionidae), Carpomyia vesuviana Costa (Diptera: Tephritidae), Bemisia tabaci Gennadius (Hemipt ra: Aleyrodidae), and Aphis gossypii Glover (Hemiptera: Aphididae) [10,[12][13][14][15]. Additionally, C. rosea is a potential biocontrol agent for several insect pests, pathogenic fungi, and nematode parasites [16].

Although some investigations on the entomopathogenic nature of C. rosea have been documented, the nutrition and media optimization for large-scale culturing remain understudied [17][18][19].The present study is also the first documentation of the entomopathogenic nature of C. rosea against the mango hopper.In addition, its efficacy as a biocontrol agent was assessed by performing an in vitro bioassay on the mango hopper.  .The samples were subjected to fungal isolation by following the Inglis et al. [20] procedure at the insect pathology laboratory, Department of Entomology, G. B. Pant University, India.Initially, the samples were soaked in distilled water for 24 h to activate the saprophytes or fungus of interest present on the surface of the cadaver.Subsequently, the cadavers were subjected to 0.3% sodium hypochlorite solution treatment to get rid of the externally grown fungi.The internally grown fungi were isolated on potato dextrose agar (PDA) media and the culture as maintained as a stock culture for further studies.


Materials and Methods


Morpho-Molecular Characterization of C. rosea

The identification of C. rosea was confirmed by morphological and molecular characterization.For the morphological study, scanning electron microscope (SEM)-based analysis was performed.For SEM analysis, the C. rosea hypha was mounted on glass slides, followed by drying and fixation using 2.5% glutaraldehyde for 2-3 h at 4 • C. The slides were washed with 0.1 M sodium phosphate buffer (7.4 pH, 3-5 min wash

The samples were dried using a KTech 850 critical
oint drier (Quorum Technologies, Lewes, UK), followed by a gold coating of the samples in a JFC 1600 gold coater (JEOL, Tokyo, Japan).The samples were finally subjected to analysis in a JSM 6610 SEM (JEOL, Tokyo, Japan).Parameters such as colony color, pigmentation of mycelium, conidiophore size, types of branches, phialide size, and size of conidia were all compared with the available literature [21][22][23][24][25][26].Further, samples were sent to the Indian Type Culture Centre, IARI, Delhi, India, for validation of the identity of the local isolates on the basis of 'phenotypic and physiological characters'.

The morphological identification of the local isolates was corroborated with molecular identification based on the internal transcribed spacer (ITS) sequencing and phylogenetic analysis.ITS1 and ITS4 regions were chosen to validate the molecular identification in this study.Genomic DNA was isolated from the fungal cultures grown on PDA by t

cetyl trimethyl ammonium bromide method [27] and further subjecte
to quality evaluation by 0.8% agarose gel electrophoresis.The PCR amplification of the ITS-rDNA region was performed using primers ITS1 and ITS4 (TCCGTAGGTGAACCTGCGG and TCCTCCGCTTATTGATATGC, respectively).The PCR was performed in a 25 µL reaction mixture using the following conditions: initial denaturation at 94 • C for 1 min followed by 35 cycles, each consisting of final denaturation at 94 • C for 30 sec, annealing temperature at 55 • C for 30 sec, initial extension for 1 min, and final extension at 72 • C for 10 min.PCR-amplified products were subjected to a 1.2%

arose gel run for con
irmation.A 1kb DNA marker was used to estimate the proper band size of amplified products and they were visualized and photographed using a Gel Doc XR system (Bio-Rad Laboratories) [28].

The 18S rDNA region was sequenced to confirm the identity of the organism.The PCR product was sequenced in both directions by using the Sanger di-de-oxy method.A search for homologous sequences was performed by BLAST analysis at NCBI (http://ncbi.nlm.nih.gov/BLAST,accessed on 13 May 2019.Multiple alignments for homology search were performed using the Clustul W algorithm software.The MEGA7 software was used to construct dendrograms of the isolate from the current study and reference sequences, retrieved from GenBank [29,30].


Media Optimization for Mass Culturing of C. rosea

Fungi were cu

A, Sabouraud's dextrose agar + 1% yeast (SDAY), Sabouraud'
maltose agar + 1% yeast (SMAY), oatmeal agar (OMA), and Czapek-Dox agar (CZA) prepared as solid media following the standard protocol.Similarly, liquid media were prepared from the same constituent excluding agar-agar.One hundred milliliters of the sterile liquid media in conical flasks of 250 mLcapacity was prepared and used throughout the experimental procedures [26].Inoculation of 0.7 cm bits of pure culture for solid and liquid media was carried out according to the study parameters such as mean radial growth, mean conidia per bit, and mean germination percentage of conidia in case of solid media and mean fresh weight or mean dry weight in case of liquid media.All these factors were further studied at 15, 20, 25, and 30 • C considering temperature as a prime and relevant factor for the vegetative and reproductive development of

ntomopathogenic fungi
[31].


Preparation of C. rosea Spore Suspension for Pathogenicity Assays

Clonostachys rosea was cultured on PDA for 15 days to develop spores on petri plates.With the help of a measuring cylinder, 10 mL of distilled water was poured onto the plates, and two drops of 0.01% Tween 80 were added.This was followed by scraping off mycelia from the plates into a suspension using a sterile infection loop.The suspension containing spores was filtered through a sterilized muslin cloth, serially diluted, and counted on a Neubauer counting chamber to achieve spore count.These spore suspensions were later used for the bioassay against A. atkinsoni.


Bioassay of C. rosea

To study the virulence of C. rosea, a bioassay on A. atkinson L. was carried out.A

insects (each treatment, 3 replications and each replicati
n had 10 insects) with four concentrations of suspensions (1 × 10 4 , 1.5 × 10 5 , 2 × 10 6 , 2.5 × 10 7 , and 3 × 10 8 spores per mL concentration) were assessed at consecutive 24,48,72,96,120,144,168,192,216, and 240 h time intervals, with control [10].Finally, the per cent mortality was calculated by IBM-SPSS v16.0 for statistical probe and Duncan's multiple range test (DMRT) at a 5% significance level.The etiology and epidemiology were studied for parameters such as the time required for adhesion, germination, colonization of fungus, first insect death, and last insect death.


Results


Collection, Isolation, and Culture Conditions of C. rosea

The isolate C. rosea 11031.19 was deposited at the Indian Type Culture Centre, IARI, Delhi, India.Further, the microscopic observation revealed a mycelium with branching hyphae and septa

that were intertwined with each other.The
ycelial growth led to the formation of round aggregates in various regions as observed with SEM (Figure 1A).The conidiophores appeared to have developed from the ropes of hyphae.The conidiophores were branched in verticillate (primary) and penicillate (secondary) forms.Phialides were flask-shaped with singular apices, unicellular, hyaline,

al, with curved-shaped conidia.The endings
f the verticillate conidiophores showed a gelatinous matrix enveloping conidia and encompassing a mass of inclusion spherical aggregates.The Olympus CX33 microscope measurements revealed that conidiophores were 83.2-95.8µm with an angle of 12.5 • -154.4 • .The size of phialides (40× objective lens) was 86.7-111.4µm with an angle for alignment in the range of 24.9 • -80.7 • .The dimensions of mycelium knots were 22.0-24.9µm.The conidial dimensions observed on SEM were in the range of 2.2-2.3 µm × 4.0-4.3µm (Figure 1B) and the hyphae h

a width of 2.0-2.3 µm.


Bioassay of
. rosea

To study the virulence of C. rosea, a bioassay on A. atkinson L. was carried out.A total of 30 insects (each treatment, 3 replications and each replication had 10 insects) with four concentrations of suspensions (1 × 10 4 , 1.5 × 10 5 , 2 × 10 6 , 2.5 × 10 7 , and 3 × 10 8 spores per mL concentration) were assessed at consecutive 24, 48, 72, 96, 120, 144, 168 , 192, 216, and 240 h time intervals, with control [10].Finally, the per cent mortality was calculated by IBM-SPSS v16.0 for statistical probe and Duncan's multiple range test (DMRT) at a 5% significance level.The etiology and epidemiology were studied for parameters such as the time required for adhesion, germination, colonization of fungus, first insect death, and last insect death.


Results


Collection, Isolation, and Culture Conditions of C. rosea

The isolate C. rosea 11031.19 was d posited at the Indian Type Culture Centre, IARI, Delhi, India.Further, the microscopic observation revealed a mycelium with branching hyphae and septate that were intertwined with each other.The mycelial growth led to the formation o

round aggregates in various regions as
observed with SEM (Figure 1A).The conidiophores appeared to have developed from the ropes of hyphae.The conidiophores were branched in verticillate (primary) and penicillate (secondary) forms.Phialides were flask-shaped with singular apices, unicellular, hyaline, oval, with curved-shaped conidia.The endings of the verticillate conidiophores showed a gelatinous matrix enveloping conidia and encompassing a mass of inclusion spherical aggregates.The Olympus CX33 microscope measurements revealed that conidiophores were 83.2-95.8μm with an angle of 12.5°-154.4°.The size of phialides (40× objective lens) was 86.7-111.4μm with an angle for alignment in the range of 24.9°-80.7°.The dimensions of mycelium knots were 22.0-24.9µ m.The conidial dimensions observed on SEM were in the range of 2.2-2.3 μm × 4.0-4.3μm (Figure 1B) and the hyphae had a width of 2.0-2.3 μm.

(A) (B)


Molecular Identification of Local Isolates

The rDNA sequence of the ITS region was estimated to be ~700 bp, confirmed with ladder specification (Figure 2a,b).Analysis with NCBI BLAST resulted in a 100% identity with C. rosea.The sequence was assigned with accession number OP673543.1.Two clades were formed on the phenogram using the consensus ITS sequence from the local isolates


Molecular Identification of Local Isolates

The rDNA sequence of the

S region was estimated to be ~700 bp, conf
rmed with ladder specification (Figure 2a,b).Analysis with NCBI BLAST resulted in a 100% identity with C. rosea.The sequence was assigned with accession number OP673543.1.Two clades were formed on the phenogram using the consensus ITS sequence from the local isolates and ten accessions were obtained from NCBI based on a maximum identity score.The local isolate ITS sequence fell in a subclade with maximum similarity with C. rosea isolated from Korea (Figure 2b).


Media Optimization for Mass Culturing

The studies of solid media at 20 • C for mean growth (Table 1) showed the highest mean radial growth in OMA, followed by SMAY, CZA, and SDAY with the lowest mean radial growth obtained in PDA.When mean spores/bit were studied at the same temperature, it was found that the most spores were obtained from OMA, followed by SDAY, CZA, and SMAY and the least production was obtained from PDA.We also evaluated mean germination percentage and found that the highest germination was obtained from OMA, followed by PDA, SDAY, and CZA with the lowest frequency seen in SMAY.After analyzing the mean radial growth, mean conidia/bit, and mean germination, the best solid media for C. rosea was OMA, followed by SDAY, CZA, and SMAY.PDA at 20 • C was found to be the least effective media in contrast with the data for the other

emperatures
used and displayed in Table 1.


Media Optimization for Mass Culturing

The studies of solid media at 20 °C for mean growth (Table 1) showed the highest mean radial growth in OMA, followed by SMAY, CZA, and SDAY with the lowest mean radial growth obtained in PDA.When mean spores/bit were studied at the same temperature, it was found that the most spores were obtained from OMA, followed by SDAY, CZA, and SMAY and the least production was obtained from PDA.We also evaluated mean germination percentage and found that the highest germination was obtained from OMA, followed by PDA, SDAY, and CZA with the lowest frequency seen in SMAY.After analyzing the mean radial growth, mean conidia/bit, and mean germination, the best solid media for C. rosea was OMA, followed by SDAY, CZA, and SMAY.PDA at 20 °C was found to be the least effective media in contrast with the data for the other temperatures used and displayed in Table 1.Similarly, at 20 • C, the highest mean fresh weight biomass was achieved (Table 2) in OMB followed by SMYB, PDB, and SDYB.The lowest mean fresh biomass was obtained from CZB.The data for dry biomass were also recorded showing the highest mean dry biomass (mycelium + conidia) obtained from OMB followed by SMYB, SDYB, and PDB.The lowest mean dry biomass was achieved in CZB.The results from the studies carried out at 15, 25, and 30 • C are pre ented in Table 2.


Efficacy of C. rosea against A. atkinsoni

The response for pathogenicity against mango hopper (Figure 3a-d) was high in two concentrations (3 × 10 8 and 2.5 × 10 7 ), with the mortality response (13.33 and 3.33%) augmenting on the 3rd day after exposure.However, lower concentrations required a longer exposure period for mortality to be achieved.At 2 × 10 6 conidia/mL and 1.5 × 10 5 conidia/mL concentrations, the mortality response (3.33 and 3.33%) started on the 4th day.For the lowest concentration of 1 × 10 4 conidia/mL, the mortality response (3.33%) started on the 5th day after exposure.On the 7th day after exposure, the mean mortality was highest at 3 × 10 8 spores/mL concentration (96.67%), followed by 2.5 × 10 7 spores/mL concentration (70.00%), and 2 × 10 6 spores/mL concentration (53.33%).However, in other lower concentrations, mortality was less than 50% (Table 3).The etiology and epidemiology of C. rosea on mango hoppers were observed by considering parameters such as the time required for adhesion, germination, colonization of fungi, first insect death, and last insect death, which were 24, 36, 48, and 192 h, respectively.Out of 30 mango hoppers, 29 were dead within 168 h.Therefore, the obtained data suggest that a colonization period of 48 h was required, whereas for complete parasitization, it would take 120-132 h.0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 3.3 ± 5.7 a 6.6 ± 5.7 a 6.6 ± 5.7 ab Control 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a Treatment response, mean values (±SE) following the same superscript letters (a, b, c, d, and e) are significantly different from each other; mean values (±SE) that are not followed by the same superscript letters (ab and bc) do not differ significantly from one another (p = 0.05%).


Discussion

Clonostachys rosea is a mitosporic-hypocreale fungus with a biotrophic nature and also a potential biological control agent against various pathogens and pests.There are reports of its isolation from various crops (barley, strawberry, rose, lettuce, cocoa, etc.), insects, nematodes, and even wine [12][13][14][15][32][33][34][35].In the present study, C. rosea was found to be a parasite of a major insect pest of mango, A. atkinsoni [2].There are very few available studies documenting the entomopathogenic nature of this fungus and, to the best of our knowledge, a study on mango hopper was never conducted.Meanwhile, this fungus has a role in controlling grey mold, crown rot, and others as a potential biocontrol agent, thus it can be considered for pest management programs [25].The majority of studies regarding C. rosea biology, media standardization, mass culture, and application for disease management, biotransformation, biodegradation, and biological energy sources have been performed considering only plant pathology applications [16,24,36].Although, to date, insects such as O. tucumana, S. grossa, H. postica, C. vesuviana, B. tabaci, Galleria mellonella, and Aphis gossypii have been studied with C. rosea, the presence of this fungus in the microhabitat of a highly remunerative crop such as mango has n

been report
d yet [10,[12][13][14][15]37].

Our data showed susceptibility of A. atkinsoni to various conidial concentrations of C. rosea (Table 3).The mortality response in the case of the highest concentration (3 × 10 8 conidia/mL) was observed on the 3rd day onwards after the exposure, wherein, the mortality recorded was found to be 13.3%.On increasing the exposure up to 7 days, 96.6% mortality was observed.This mortality response is consistent with the study conducted by Prabhu and Kumar [12], where a concentration of 10.52 × 10 6 spores/mL caused mortality of A. stephensi (92.8%) and C. quinquefasicatus (71.9%) in 72 h.Similarly, Mustafa et al. [13] from the northern region of Iraq reported 55.1% mortality of alfalfa weevil (Hypera