Entomopathogenic Potential of Simplicillium lanosoniveum Native Strain in Suppressing Invasive Whitefly, Aleurodicus rugioperculatus Martin (Hemiptera: Aleyrodidae), Infesting Coconut

In 2016, infestation of an exotic polyphagous pest, the rugose spiraling whitefly (RSW), Aleurodicus rugioperculatus Martin (Hemiptera: Aleyrodidae), was documented on coconut for the first time in India. Instantaneously, RSW has garnered wide attention owing to its damage severity and rapid spread across the coconut-growing regions of the country. Hence, an attempt was made to devise a sustainable integrated pest management (IPM) module using biological control agents as a mainstay component. The present study documented the identification and characterization of a potential entomopathogenic fungal isolate for the management of RSW. An entomopathogenic fungus isolated from nymphal cadavers of RSW was identified as Simplicillium lanosoniveum based on morphological and phylogenetic analyses. A gradient of five conidial concentrations (1 × 104, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 conidia/mL) of the S. lanosoniveum were tested against eggs, first instars, second to third instars and pupae of RSW. Results revealed that S. lanosoniveum is highly virulent to all developmental stages of RSW by causing mortality rates of 95.20%, 87.33%, 85.38% and 72.85%, in eggs, initial, middle and later instar nymphs of RSW, respectively, at the highest tested concentration (1 × 108 conidia/mL) at seven days after exposure. The LC50 and LT50 values of S. lanosoniveum were 4.72 × 104, 4.94 × 104, 5.11 × 105, 5.92 × 105 conidia/mL and 4.27, 4.86, 4.56, 5.89 days against eggs, initial, middle and later instar nymphs of RSW, respectively. Further, preliminary field trials with S. lanosoniveum strain at 1 × 108 conidia/mL exhibited a significant reduction in the egg and nymphal population by 57.8% and 56.3%, respectively. This report thus demonstrated that the newly isolated S. lanosoniveum is an effective pathogen at suppressing all the developmental stages of RSW. This is the first record of S. lanosoniveum infecting RSW, and it has a great potential to be developed as a mycoinsecticide.


Introduction
Rugose Spiraling Whitefly (RSW), Aleurodicus rugioperculatus Martin (Hemiptera: Aleyrodidae) is an introduced pest, whose infestation on coconut was reported for the first time from Pollachi, Tamil Nadu (10.6609 • N, 77.0048 • E) and Palakkad, Kerala (10.7867 • N, 76.6548 • E) state in India [1]. RSW infestation on coconut was first described from Belize [2], and Central America is believed to be the centre of origin for the pest. RSW is a highly polyphagaous pest with more than 118 hosts belonging to 43 diverse plant

Insect Cultures
RSW adults were collected from infested coconut gardens of the research farm belonging to ICAR-Central Plantation Crops Research Institute (ICAR-CPCRI), Kasaragod, Kerala, India (12 • 30 N latitude, 75 • 00 E longitude and 10.7 m). RSW culture was reared on coconut seedlings raised in pots placed in the greenhouse with natural photoperiodic conditions. Different developmental stages of RSW such as eggs, first instar, second instar, third instar and fourth instar nymphs (pupae) were collected from the reared culture and were used for bioassay studies.

Fungal Isolation
RSW nymphal cadavers (n = 4) fully covered with fungal mycelia under natural growth conditions were collected from a coconut garden at ICAR-CPCRI, Kasaragod (12 • 30 N latitude, 75 • 00 E longitude and 10.7 m). Sterile needles were used to isolate mycelia or conidia from the cuticles of nymphal cadavers transferred to a Petri plate containing Potato Dextrose Agar (PDA) medium and cultured at 26 ± 1 • C and 60% RH for 7-10 days in a biochemical incubator (BOD). In order to isolate the pure culture, the hyphal tip from a small fungal colony was transferred to PDA slants and incubated at 26 ± 1 • C and 60% RH. The morpho-taxonomic characteristics of the fungus were identified based on conidia-forming mycelia and conidial structure [15,16].

Identification of the Fungus 2.3.1. Morphological Identification
Morphological identification of the fungal isolate was recorded on PDA medium incubated at 26 ± 1 • C and 60% RH for seven to ten days. The micro-morphological features were recorded under a compound microscope (Nikon Eclipse 80i) attached with a digital camera. The identification of the fungus was further confirmed with the help of Fungal Identification Services, Agharkar Research Institute (ARI), Pune, Maharashtra, India.

Genetic Identification
Total genomic DNA was extracted from five-day old single spore cultures of fungus grown in 100 mL Potato Dextrose broth. After vacuum filtration, the mycelia were washed with sterilized distilled water and powdered using liquid nitrogen with the help of a mortar and pestle. The DNA was extracted from powdered preparations using DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) following the standard manufacturer's instructions. The fungal ITS region was amplified using the primers: ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) and ITS6 (5 -GGAAGTAAAAGTCGTAACAAGG-3 ) [17]. Two more nuclear genes viz., elongation factor-1 alpha (TEF1-α) and RNA polymerase II largest subunit 1 (RPB1) of the ribosomal RNA gene were used in addition to confirm the identification the species level. The DNA was amplified using specific primers of the TEF1-α (983F 5 GCYCCYGGHCAY-CGTGAYTTYAT 3 2218R 5 ATGACACCRACRGCRACRGTYTG and RPB1 (CRPB1 5 CCWGGYTTYATCAAGAARGT 3 and CRPB1 A 5 CAYCCWGGYTTYATCAAGAA 3 RPB1Cr 5 CCNGCDATNTCRTTRTCCATRTA 3 [18]. The resultant PCR products were excised and purified using a Qiagen Gel Extraction Kit (Qiagen India) following the standard protocol and were sequenced at Agrigenome, Cochin, India. The generated sequences were submitted to NCBI GenBank (Accession numbers: ITS = MK463992, TEF1-α = MW893683 and RPB1 = MW836952) and used for multi gene phylogenetic analysis. The sequences were used as query to perform BLAST search (http://blast.ncbi.nlm.nih.gov/ (accessed on 8 April 2021), and the sequence similarity studies were performed using ClustalX (1.81). The phylogenetic tree of S. lanosoniveum was constructed following maximum likelihood (ML) method using MEGA X software [19].

Conidial Preparation
The fungal culture was inoculated in Potato Dextrose Broth (PDB) and incubated at 26 ± 1 • C and 60% RH on a shaker for 14 days. The mycelia mat along with conidia was thoroughly mixed in a mixer grinder, and the suspension was filtered through three layers of muslin cloth, in order to get a hyphal-free conidial suspension. Conidial suspensions were prepared in sterile distilled water containing 0.01% Tween 80 and the concentration of the suspension was adjusted to different conidial concentrations using Neubauer's improved haemocytometer under a Nikon eclipse 80i compound microscope. Five different spore concentrations ranging from 1 × 10 4 to 1 × 10 8 conidial concentration/mL were tested against different developmental stages of RSW.

Experimental Design
Coconut leaf bits containing 35-40 different developmental stages of RSW were collected from the greenhouse culture. Thirty leaf bits (10 cm each) were gently dipped in freshly prepared respective conidial suspensions for 20 s and placed separately on a Petri plate (14 × 2.5 cm) over sterilized wet tissue paper. Sterile distilled water containing 0.01% Tween 80 alone was used as the control. The experiment had five replications for each concentration, and the whole bioassay was repeated twice. Prior to conidial spray, the number of various developmental stages of RSW on each coconut leaf bit was counted to maintain a uniform population across the treatments. The mortality data were recorded by counting dead cadavers and developmental stages with fungal infection at 24 h intervals up to seven days, and the data was subjected to Abbott's formula [20] for calculating corrected mortality. For time mortality studies, the higher concentration at 1 × 10 8 spores/mL were used to determine LT 50 of S. lanosoniveum against RSW developmental stages.

Field Virulence of S. lanosoniveum
Two field trials using S. lanosoniveum carried out during November 2019 and January 2020, respectively, in the RSW infested coconut gardens of ICAR-CPCRI, Kasaragod, Kerala (average temperature 22-33 • C, 60-73% RH during December 2019 and average temperature 20.6-32.4 • C, 59-71% RH during January 2020, respectively). The conidial suspension of the fungal culture (1 × 10 8 conidia/mL) was prepared in sterile aqueous solution containing 0.01% Tween 80. The spore suspension of 500 mL was sprayed thoroughly on the dwarf cultivars of each coconut tree (variety; Chowghat Orange Dwarf (COD), n = 20) which were naturally infested with different developmental stages of RSW through handheld sprayer. Prior to fungal spray, the numbers of developmental stages of RSW per leaflet (5 leaflets per palm) were counted under a Nikon Stereozoom microscope (SMZ 800 N). Twenty dwarf palms sprayed with sterile aqueous solution containing 0.01% Tween 80 were maintained as control. Mortality data ware recorded from the treated and control plants after 14 days of spray. The experiment was replicated thrice and field bioassays were conducted twice at different time intervals. Developmental stages with S. lanosoniveum fungal infection recorded using a Nikon Stereozoom microscope (SMZ 800 N) and the percent mortality was calculated.

Statistical Analysis
Conidial concentration, time and their interactive effects on mortality per cent data were analyzed using PROC GLM (SAS version 9.3, SAS institute, 2011). Mortality data of different developmental stages of A. rugioperculatus were corrected using Abbott's formula [20]. The treatment difference was evaluated using least significant difference (LSD) at p < 0.05. The dose and time dependent mortality studies to kill 50% of the population (LC 50 and LT 50 ) were calculated by probit analysis [21] using R software based on binomial GLM function [22,23]. Field efficacy data were assessed by Student's t-test with a significance of difference at p < 0.01 [24].

Morphological Identification
Based on the morphological characters, the fungus isolated from the field collected nymphal cadavers of A. rugioperculatus was identified as Simplicillium lanosoniveum (Cordycipitaceae: Hypocreales). The fungal colonies were characterized on PDA medium as white, velvety with radial cracks and primrose yellow on the reverse side ( Figure 1). Conidia was (2-4 × 1-2 µm), oval ellipsoidal, hyaline, smooth walled, adhering in globose to ellipsoidal head at the apex of phialides ( Figure 1).

Genetic Identification
The sequences of Simplicillium isolate generated in the present study have been deposited in GenBank with accession numbers: ITS = MK463992, TEF1-α = MW836952 and RPB1 = MW893683. BLAST analysis of generated sequences exhibited 99% sequence similarity with Simplicillium lanosoniveum (DQ522406 and DQ522357). A phylogenetic tree of Simplicillium at species level was generated using maximum-likelihood (ML) analysis based on a combined data set of ITS, TEF1-α and RPB1. Multigene phylogenetic analysis showed that Simplicillium isolate (MK463992, MW836952 and MW893683) of the present study have a close phylogenetic affinity to Simplicillium lanosoniveum with strong bootstrap support (98-100%, Figure 2).

Genetic Identification
The sequences of Simplicillium isolate generated in the present study have been deposited in GenBank with accession numbers: ITS = MK463992, TEF1-α = MW836952 and RPB1 = MW893683. BLAST analysis of generated sequences exhibited 99% sequence similarity with Simplicillium lanosoniveum (DQ522406 and DQ522357). A phylogenetic tree of Simplicillium at species level was generated using maximum-likelihood (ML) analysis based on a combined data set of ITS, TEF1-α and RPB1. Multigene phylogenetic analysis showed that Simplicillium isolate (MK463992, MW836952 and MW893683) of the present study have a close phylogenetic affinity to Simplicillium lanosoniveum with strong bootstrap support (98-100%, Figure 2).

Genetic Identification
The sequences of Simplicillium isolate generated in the present study have been deposited in GenBank with accession numbers: ITS = MK463992, TEF1-α = MW836952 and RPB1 = MW893683. BLAST analysis of generated sequences exhibited 99% sequence similarity with Simplicillium lanosoniveum (DQ522406 and DQ522357). A phylogenetic tree of Simplicillium at species level was generated using maximum-likelihood (ML) analysis based on a combined data set of ITS, TEF1-α and RPB1. Multigene phylogenetic analysis showed that Simplicillium isolate (MK463992, MW836952 and MW893683) of the present study have a close phylogenetic affinity to Simplicillium lanosoniveum with strong bootstrap support (98-100%, Figure 2).

Virulence Bioassay
The pathogenicity of S. lanosoniveum was evaluated against different developmental stages of RSW. The fungal bioassay treatment resulted in the highest pathogenicity to the eggs, initial (first), middle (second-third) and later (fourth) instar nymphs of RSW with corrected mortality rates of 95.20%, 87.33%, 85.38% and 72.85%, respectively following the seventh day after treatment (DAT) at 1 × 10 8 conidial concentrations/mL (Figure 3).

Determination of the Lethal Concentration (LC50) and Lethal Time (LT50)
Significant differences in the LC50 and LT50 values of S. lanosoniveum against different life stages of A. rugioperculatus were documented based on the dose-and time mortality response studies (

Determination of the Lethal Concentration (LC 50 ) and Lethal Time (LT 50 )
Significant differences in the LC 50

Field Virulence of Entomopathogenic Fungi
Fourteen days after the spray of S. lanosoniveum, the mortality of eggs and nymphal stages of RSW was calculated. As shown in Figure 5, the population of eggs (t = 4.56; p < 0.01) and nymphs (t = 8.19; p < 0.01) of A. rugioperculatus were reduced by 57.8% and 56.3%, respectively at 14 DAT. Thus, the native fungi isolated and reported herein showed positive virulence by infecting RSW stages under open field conditions ( Figure 5).

Discussion
The first incidence of the RSW was identified in India during the year 2016 following which it has spread widely and caused serious damages in larger areas of crop cultivation, particularly in coconut [6][7][8]. The adoption of management strategies using chemical pesticides is uneconomical and it also causes severe adverse effects on naturally existing biocontrol agents including Encarsia guadeloupae Viggiani (Hymenoptera: Aphelinidae) [7,8]. The conservation of natural enemy complexes and avoidance of insecticide spray are the major pest management strategies provided to the farmer's against this invasive pest to avoid the ill effects of pesticide usage. In this context, utilization of entomopathogenic fungi as a means for biological control of RSW infestations is of significant importance [3][4][5][6].
However, exploration and identification of novel and virulent EPF strains adapted to particular localities is necessary for the development of effective mycoinsecticides. Efficacy of entomopathogenic fungi for managing the sucking insect pest population in the field conditions is well recognized [25] owing to their unique mode of action of penetrating the insect cuticle [26,27]. Among the EPFs, Akanthomyces species are widely utilized as commercial biological pesticides against whiteflies, thrips and aphids [28]. Fungicolous and entomopathogenic species of Akanthomyces were later placed in the genera Simplicillium and Lecanicillium based on systematic studies [16]. Although species of Simplicillium occurs in a broad range of ecological niches [16,[29][30][31][32] but only few studies have reported the pathogenicity of Simplicillium sp. against insects [33][34][35][36][37][38][39][40]. In the present study, we report the identification of a novel isolate of entomopathogenic fungus, S. lanosoniveum isolated from RSW in the invaded region. Bioassay studies have revealed the potency of the isolate S. lanosoniveum, as it was found to be virulent against all the developmental stages of RSW. The ecological and economical value of Simplicillium sp. for its biocontrol and bioactive features were highlighted by many researchers [41][42][43]. S. lanosoniveum is a known phytopathogen, causing brown spots and lesions on flowers [44] or as a mycoparasite on soybean rust [45,46]. This species is also reported as a pathogen of aphids [33] and as an anti-Trichomonas vaginalis agent [47]. Other species of Simplicillium such as S. chinense acts as a biological control agent against plant parasitic nematodes [39,48]. S. lamellicola is known to suppress plant bacterial diseases and grey mould of tomato and ginseng [31,49]. Bioactive compounds with anti-fungal and anti-bacterial profiles and pharmaceutical exopolysaccharides were isolated from S. lanosoniveum [50][51][52][53]. Linear and cyclic peptides with anti-fungal, anti-bacterial and anti-viral properties were also discovered from the secondary metabolites of S. lamellicola and S. obclavatum [31,54]. The entomopathogenity of S. lanosoniveum was demonstrated against silkworms, and it was evident that virulence of the isolate was strong as that of B. bassiana [38]. The present study strain, S. longisoniveum, exhibited >70-80% mortality rates against RSW nymphs and pupae, while >90% mortality recorded for egg stage at 1 × 10 7 and 1 × 10 8 conidia/mL under laboratory bioassays on seven DAT. It was noticed that early instars are highly susceptible to this fungal strain than the late instars. Present study results are corroborating the earlier report of Nagasi et al. [55], where, B. bassiana was found most virulent against the first instar of silver leaf whitefly, Bemisia argentifolii (Bellows & Perring). Entomopathogenic fungi used as an effective biological control against in other whitefly species like, B. argentifolii and Trialeurodes vaporariorum (Westwood) [55][56][57]. Eyal et al. [58] reported that B. bassiana caused 52-98% mortality in Bemisia tabaci (Gennadius) at concentrations of 1-4 × 10 6 conidia/mL. The present study observed increased mortality of different developmental stages of RSW with an increase in the conidial concentration and time of exposure. Likewise, Boopathi et al. [59] also observed that the application of I. fumosorosea was highly pathogenic to A. dispersus on cassava and exhibited differences in efficacy between 3 and 15 days after treatment. In the present study, the LC 50 values and LT 50 values of S. lanosoniveum against eggs, first instar, second to third instar, and pupae of RSW were 4.72 × 10 4 , 4.94 × 10 4 , 5.11 × 10 5 , 5.92 × 10 5 conidia/mL and 4.27, 4.86, 4.56, 5.89 days, respectively. Similarly to present results, the LC 50 values of I. fumosorosea for eggs, first-second, third and fourth instar nymphs of RSW were estimated to be 2.6 ×10 4 , 2.3 × 10 4 , 3.5 × 10 5 and 9.5 × 10 5 spores /mL respectively and the LT 50 values were4.19, 3.44, 5.62 and 5.69, respectively for corresponding life stages for the concentration of 1 × 10 8 conidia/mL tested [60]. Therefore, in terms of the pathogenicity bioassays, the LC 50 and LT 50 values of S. lanosoniveum exhibited significant biocontrol potential in the management of RSW under laboratory conditions. The results are comparable to the effects produced by the widely studied entomopathogenic fungi like I. fumosorosea [60]. However, S. longisoniveum at 1 × 10 8 conidia/mL tested under field conditions caused 57.8% and 56.3% mortality in the egg and nymphal population of RSW on 14 DAT, which was less than that of laboratory efficacy. The reason for this reduction in efficacy in the field condition might be due to the involvement of abiotic factors, as field conditions are heterogenetic in nature compared to the homogenic environment of the laboratory. Earlier reports also suggested that environmental factors not only affect survival and virulence of entomopathogenic fungi, but also influence their host-pathogen interaction [61]. Temperature and relative humidity are the two most important environmental factors which greatly influence the fungal germination, infection, sporulation, survival and virulence of the entomopathogenic fungi [62][63][64]. In addition, the process of fungal infection is also governed by the microclimate of the insect cuticle [65]. However, the sensitivity of the isolate or specific strain varies with environmental factors [62] and the success of any entomopathogenic fungi is determined by its adaptability to the local or prevailing environmental conditions [66]. Therefore, it is imperative to ascertain the effect of temperature and RH on any new isolate before considering it for biocontrol of insect pests. The fungal isolate reported herein performed fairly well in reducing the whitefly population in the open field conditions where the prevailing temperature and relative humidity conditions could have played a crucial role in affecting its efficacy. To conclude, this is the first report of natural occurrence of S. lanosoniveum on RSW. This study identified a highly pathogenic fungal isolate, S. lanosoniveum, based on its morphology, molecular and phylogeny and demonstrated its pathogenicity against RSW under in vitro and field conditions. Hence, this study forms a foundation for the large scale development of a biocontrol agent to suppress the spread of RSW. However, further studies on its compatibility with natural enemies for field applications are required. Efforts are also in progress to develop formulations with high virulent stability to sustain its field efficacy.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.