Identification of locally isolated entomopathogenic Fusarium species from the soil of Changa Manga Forest, Pakistan and evaluation of their larvicidal efficacy against Aedes aegypti

Abstract Dengue fever vectored by the mosquito Aedes aegypti is one of the most rapidly spreading insect-borne diseases. Current reliance of dengue vector control is mostly on chemical insecticides. Growing insecticide resistance in the primary mosquito vector, Aedes aegypti, limits the effectiveness of vector control through chemical insecticides. These chemical insecticides also have negative environmental impacts on animals, plants and human health. Myco-biocontrol agents are naturally occurring organisms and are found to be less damaging to the environment as compared to chemical insecticides. In the present study, entomopathogenic potential of local strains of fungi isolated from soil was assessed for the control of dengue vector. Local fungal isolates presents better alternative to introducing a foreign biocontrol strain, as they may be better adapted to environmental conditions of the area to survive and may have more entomopathogenic efficacy against target organism. Larvicidal efficacy of Fusarium equiseti and Fusarium proliferatum was evaluated against Aedes aegypti. Local strains of F. equiseti (MK371718) and F. proliferatum (MK371715) were isolated from the soil of Changa Manga Forest, Pakistan by using insect bait method. Larvicidal activity of two Fusarium spp. was tested against forth instar larvae of A. aegypti in the laboratory, using concentrations 105, 106, 107 and 108 conidia /ml. LC50 values for F. equiseti after 24h, 48h, 72h and 96h of exposure were recorded as 3.8x 108, 2.9x107, 2.0x107, and 7.1x106 conidia /ml respectively while LC50 values for F. proliferatum were recorded as 1.21x108, 9.6x107, 4.2x107, 2.6x107 conidia /ml respectively after 24h, 48h, 72h and 96h of exposure. The results indicate that among two fungal strains F. equiseti was found to be more effective in terms of its larvicidal activity than F. proliferatum against larvae of A. aegypti.


Identification of locally isolated entomopathogenic Fusarium species from the soil of Changa Manga Forest, Pakistan and evaluation of their larvicidal efficacy against Aedes aegypti
Pakistan and to evaluate the larvicidal efficacy of these isolates against larvae of Aedes aegypti.

Isolation of F. equiseti and F. prolifertum
Local strains of Entomopathogenic F. equiseti and F .proliferatum were isolated from Changa Manga Forest, Pakistan. Changa Manga (31 o 05`01.9" N and 73 o 59`45.1"E) is a forested area located 60 km away from Lahore within the District of Kasur. The total area is 12510 hectors approximately. The soil of the Changa Manga Forest is highly porous, dark brown in color with silty loam texture. The soil of the forest is considered to be healthy and fertile for the plantation of trees and supports a variety of soil born fungi. The pH of soil ranges from 7.7 to 8.0. Isolation of fungi from soil samples collected from Changa Manga Forest was attempted using the Galleria bait method (Meyling and Eilenberg, 2006). Final instars of wax moth (Galleria mellonella) were used as bait insects. Wax moth larvae were surface-sterilized using 1% sodium hypochlorite and rinsing them in distilled water, and incubated at 25°C. 1g of Soil sample sprinkled on PDA petri plates with 10 larvae of wax moth in each soil sample was used for baiting. Samples were observed after 7 days to check any fungal growth for the first time and on daily basis after initial seven days. Colonies of Fusarium species found in soil samples were isolated and subcultures on PDA plates to obtain the fungal strain on separate plate. The pure cultures of fungal isolates were mass cultured PDA plates.

Identification and phylogenetic analysis of fungal isolates
Isolated Fusarium spp. were identified on basis of morphological traits using taxonomic key (Summerell et al., 2003;Nayyar et al., 2018) and with the help of experts. For DNA extraction fungal mycelia was obtained after 10 min of centrifugation at 17500 rpm and 300 μl of extraction buffer (200mM Tris-HCl, pH 8.5, 250mM NaCl, 25mM EDTA, 0.5% SDS) was added in culture media. The suspension was vortexed with 2.8 mm stainless steel beads during 10 minutes. After centrifugation at 17500 rpm for 10 min, 150 μl of 3M sodium acetate (pH 5.2) was added to the supernatant. The supernatant was incubated at -20ºC for 10 more minutes and centrifuged (17500 rpm, 10 min). The DNA-containing supernatant was transferred to a new tube and nucleic acids were precipitated by adding 1 volume of isopropyl alcohol. After 5 minutes of incubation at room temperature the DNA suspension was centrifuged (17500 rpm, 10 min). The (DNA) pellets were washed with 70% ethanol to remove residual salts. Finally, the pellets were air-dried and the DNA was re-suspended in 50 μl of buffer (10mM Tris-HCl (pH 8), 1mM EDTA).
Specific PCR assays were carried out using primers NS1 5' (GTA GTC ATA TGC TTG TCT C) 3' and NS8 5' (TCC GCA GGT TCA CCT ACG GA) 3' (melting point 42 ºC) for identification of fungal spp.. The PCR purified products were directly sequenced by the company Macrogen (Seoul, Korea). Blast n-searches in GenBank permitted the identification of the level of identity with known sequences, and subsequently the spp. corresponding to the isolates. The sequences generated from 18S rRNA clustered with similar taxa. Phylogenetic analysis was done with the maximum likelihood algorithm and general time-reversible model

Introduction
Dengue is one of the most prominent vector-borne diseases in Pakistan. Its incidence is emerging despite the implementation of routine national dengue control program. The potential vector and supporting environmental conditions which promote the infectious agent to proliferate are playing important role in spreading dengue. The etiological agent for dengue is arbovirus and the major vector responsible to transmit the disease is A. aegypti (Heinisch et al., 2019).
Application of chemical insecticides is the widely used control method for dengue vector, which heavily contaminate the environment and negatively affect humans and other organisms. Therefore scientists are searching for safer alternative methods of dengue vector control (Nassar, 2018). Biological control is a promising and potentially effective replacement to chemical insecticides. It employs the deliberate use of microorganisms for regulating the population of dengue vector and preventing the transmission of disease (Hajek and Eilenberg, 2018). Entomopathogenic fungi are among the wide spread group of microorganisms which are used to provide biocontrol against insects (Jaber and Ownley, 2018). Entomopathogenic fungi are natural foes of insects with cosmopolitan existence and rich diversity, presenting a sustainable solution for dengue vector control (Antonio et al., 2001, Araujo andHughes, 2016). These fungi are pathogenic to insects and due to their eco-friendliness these are preferred to kill insects at various stages of insect life cycle (Shang et al., 2015).
Various strains of fungi present effective control against disease spreading insects. Laboratory bioassays provide basis for the control of disease causing insects in field. In many research studies, the entomopathogenic fungi are reported to be an effective biological control tool against various insect spp. (Feng et al., 1994;Shah and Pell, 2003;Charnley and Collins, 2007;Araujo and Hughes, 2016;Lovett et al., 2019). The current study lies in accordance with previous studies using entomopathogenic fungi for control of Aedes aegypti (Dong et al., 2012;Evans et al., 2018;Vasantha-Srinivasan et al., 2019). Fusarium is a large genus of filamentous fungi abundantly found in soil, associated with both living and dead animals. Among animals most of the Fusarium spp. are primarily found in association with insects. Some species of Fusarium are found to be strong entomopathogens of members of order dipteran and homopteran in laboratory bioassays and field observations. Most of these spp. show high host specificity and minimal damage to non target organisms (Teetor-Barsch and Roberts, 1983;Brimner and Boland, 2003;Dutta, 2015.). Fusarium equiseti and Fusarium proliferatum are reported to be entomopathogens of many insect species including Tribolium confusum, Galleria mellonella, Cephus cinctus, Brahmina coriacea etc. according to various research studies (Al-Ani et al., 2018, Kalaimurugan et al., 2019Santos et al., 2020;Akhter et al., 2021). Entomopathogenic fungi are widely distributed in a wide range of terrestrial ecosystems including forest, agricultural and pasture habitats (Pell et al., 2001, Nidhi et al., 2018. Changa Manga forest is a man-made forest in Pakistan. The soil properties of this forest serve an ideal habitat for a vast variety of entomopathogenic fungi (Abrar et al., 2020). Objectives of the present research are to identify the local strains of Fusarium spp. isolated from soil of Changa Manga forest, of sequences evolution using the model testing feature of MEGA6 software (Tamura et al., 2011). Bootstrap consensus trees were inferred from 1000 replicates, and corresponding bootstrap values >50% are cited in the trees.

Rearing of Aedes aegypti
Aedes mosquitoes were collected from public parks of Lahore, Pakistan. Aedes mosquitoes were morphologically identified according to guidelines by Rueda (2004) under the supervision of parasitology experts. The Aedes mosquito rearing was done at Laboratory of Parasitology Department, University of Veterinary Sciences, Lahore where rearing conditions were simulated. Temperature and relative humidity was set at 27°C ± 2°C & 75% ± 5% respectively. For the successful rearing of mosquitoes, temperature and humidity were maintained at the particular required levels. The humidity of the cages was maintained with the help of wet cotton draped over them. The development and various stages in the life cycle of the mosquito are affected by photoperiod and light intensity. For the uniform development of A. aegypti mosquitoes a cycle of 14 hours of light and 10 hours of darkness was maintained (Rund et al., 2020).

Larvicidal bioassay
The conidial suspension of fungal isolates was prepared. 0.01% tween80 solution was prepared by mixing 0.1 ml of tween80 in 1000 ml of distilled water in conical flask. About 20 ml of 0.01% Tween80 was poured in the plate containing fungal colony and swirled handily and gently to favor the detachment of conidia. The procedure was repeated for 10-15 plates of each fungal strain to obtain the suspension solution. All the prepared solutions were stirred with magnetic stirrer (IRMECO, Germany) to ensure complete mixing, and then stored at 4°C for further use. The number of conidia per ml of fungal strains was counted using haemocytometer (Marienfeld Superior, Germany) under the microscope (NIKON DS-L2, Japan) to prepare the dilutions of 10 5 , 10 6 , 10 7 and 10 8 conidia per ml respectively, as per requirement of experimentation. 0.01% Tween 80 was used as blank control. Conidial suspension obtained was transferred to conical flask. The larval mortality bioassays were carried out according to the standard method (WHO, 2005). Batches of 20 fourth instar larvae were exposed to a range final concentration of 10 5 , 10 6 , 10 7 and 10 8 conidia/ml in polyethylene cups. All cups were covered with net sheets. 20 fourth instar larvae in water without conidial suspension served as the control group. The larval mortality was recorded after 24 h, 48 h, 72 h and 96h. Laboratory conditions were similar to rearing conditions. Three replicates of larvicidal bioassays were carried out for the study.

Statistical analysis
The larval mortality was calculated using the following formula The data on the efficacy were subjected to probit analysis (Finney, 1971;Waris et al., 2020) and LC 50 values were calculated using SPSS version 16.0.

Identification and phylogenetic analysis of fungal isolates
Morphological and molecular identification of the local strains confirmed the Fusarium spp. as Fusarium equiseti and Fusarium proliferatum.

Macroscopic and microscopic identification
Colony morphology of F. equiseti showed white cotton mycelium with velvety texture and peach undersurface. The Growth was rapid. Long slender macroconidia born on aerial mycelium with generally 5-7 septate and 15 to 30μm in length were present while microconidia were absent. Hyphae were septate and hyaline and chlamydospores were formed in chains (Figure 1). In case of F. proliferatum white villous colonies were observed with moderate to rapid growth and cottony texture. A large number of macroconidia of about 10-25μm in length with 3-5 septa and slender mycelium. A large number of slender microconidia with no septa and 5-15x 1.3 to 5.5 μm in size were present. Chlamydospore was absent (Figure 2).

Molecular characterization and phylogenetic analysis of Fusarium spp.
Three sequences of ITS region of Fusarium spp. were amplified resulted in consensus sequence of up to 1700bp. Initial BLAST (Basic Local Alignment Tool) search of the consensus of locally isolated Fusarium equiseti (MK371718) showed 99% similarity with Chinese sequence of F. equiseti (KJ413063) with query cover of 98%, while isolated strain of F. Proliferatum (MK371715) showed 99% identity with F. proliferatum (LT841264.1 & LT841250.1) of Netherlands with query cover 99%. Closely related spp. were retrieved from Genbank and sequences of published work were also put in final dataset. Sequences were aligned using MUSCLE online alignment tool. Missing base pairs were corrected in BioEdit. Maximum Likelihood phylogram was constructed in MEGA 6 using Kimura 2-parameter model with Gamma distributed sites. Final dataset retrieved after phylogeny was made up to 23 sequences. Total characters of this analysis are 1088 among them 404 were conserved, 583 variable, 322 were parsimony and 260 were singleton sites. Two sequences produced during this study shows similarity with F. equiseti and F. Proliferatum (Figure 3).

Entomopathogenic efficacy of locally isolated Fusarium spp.
Entomopathogenic efficacy of two Fusarium spp. was evaluted against A. aegypti to be utilized in promoting biological control technologies. A number of researchers have concluded Fusarium spp. as efficient entomopathogen against many insects including Culex quinquefsciatus, Anopheles stephensi, Sitophilus oryzae, Tetranychus neocaledonicus, Tropidacris collaris, Lymantria obfuscate, Tetranychus urticae etc. (Munshi et al., 2008;Mohanty et al., 2008;Rachana et al., 2009;Pelizza et al., 2011;Batta, 2012;Tangtrakulwanich et al., 2014;Vivekanandhan et al., 2018;Wu et al., 2018). Santos et al. (2020) published a review study based on a survey of peer-reviewed papers published from 2000 to 2019, presenting the significant pathogenicity of Fusarium spp. against various insects. This survey was made using search strings in a number of databases. According to the review survey at least 30 spp. and 273 isolates of Fusarium were reported as pathogenic to at least one spp. of insect. Ten complexes of Fusarium spp. harbor entomopathogenic fungi, of which F. equiseti, F. fujikuroi, F. oxysporum and F. solani spp. complexes represented the most abundant number of entomopathogenic strains.
The present study is focused on the entomopathogenic potential of locally isolated strains of Fusarium equiseti and Fusarium proliferatum from soil of forested areas of Pakistan against Aedes aegypti. Local fungal isolates offer a superior alternative to introducing a foreign biocontrol strain, as they may be better adapted to both kill local mosquitoes and survive local conditions. There are also regulatory and ecological advantages to using strains already present in the country or in the ecosystem (Bilgo et al., 2018). Results of the present study showed that both isolates were effective against A. aegypti population (Table 1). In case of F. equiseti the highest mortality percentage of larvae was recorded as 91% after exposure to 1x10 8 conidia/ml dose treatments in 96 h. 10 5, 10 6 , 10 7 and 10 8 conidia /ml concentration revealed positive relationship of F. equiseti against A. aegypti. LC 50 values for F. equiseti after 24h, 48h, 72h and 96h of exposure were recorded as 3.8x 10 8 , 2.9x10 7 , 2.0x10 7 , and 7.1x10 6 conidia /ml respectively. Entomopathogenic outcome of conidial suspensions of F. proliferatum against larvae of A. aegypti was assessed and maximum mortality was observed after exposure of 96h (82%). LC 50 values for F. proliferatum were 1.21x10 8 , 9.6x10 7 , 4.2x10 7, 2.6x10 7 conidia /ml 1.21x10 8 , 9.6x10 7 , 4.2x10 7 , 2.6x10 7 conidia/ml respectively after 24h, 48h, 72h and 96h of exposure. It was found that mortality percentage of larvae increased with the increase in time and conidial concentration of these entomopathogenic fungi. F. equiseti (MK371718) was found to be more effective than F. Proliferatum (MK371715) against larvae of A. aegypti. No mortality was observed in the control group. Results of present study showed that Local isolates of F. Equiseti and F. Proliferatum were found to be effective against larvae of Aedes aegypti. But taking long time to kill 50% population when compare with the synthetic insecticides, is the only drawback for the use of these entomopathogenic fungi. The present research study is in agreement with the study conducted by Bilal et al. (2012) which showed the same trend. They isolated the entomopathogenic fungus Metarhizium anisopliae from the local environment, and evaluate its efficacy against the suspected dengue vector Aedes albopictus in Pakistan and the results demonstrated that M. anisopliae had larvicidal effect with LC 50 value 1.09×10 5 and LC 90 value 1.90×10 13 while it took 45.41 h to kill 50% of tested mosquito population.
The results of the present study indicated the potentiality of the two fungal isolates Fusarium equiseti and Fusarium proliferatum for use in control of Aedes aegypti populations in laboratory conditions. Conidial suspensions of Fusarium equiseti were found to be more effective against Aedes aegypti than Fusarium proliferatum. However, further research can be performed to determine the entomopathogenic efficacy of these fungal strains by conducting field trials.