Morphological and molecular identification of Trichoderma asperellum isolated from a dragon fruit farm in the southern Philippines and its pathogenicity against the larvae of the super worm, Zophobas morio (Fabricius, 1776) (Coleoptera: Tenebrionidae)

Trichoderma asperellum (Ascomycota: Hypocreaceae) is a globally recognized soil fungus due to its broad-spectrum antimicrobial and plant growth-promoting properties. To increase the availability of local strains, soil samples from a dragon fruit farm in Villanueva (Misamis Oriental, Philippines) were collected and baited using the insect-baiting technique. Using the baiting technique, T. asperellum strain, RMCK01, was isolated. The strain was characterized using morphological and molecular data and its biological control potential was tested using different conidial concentrations on the last larval instar of the super worm, Zophobas morio (Fabricius, 1776) (Coleoptera: Tenebrionidae). The ITS1 sequences of T. asperellum RMCK01 were 100% identical to the sequences of other T. asperellum isolates reported from Vietnam, India, Thailand, and China. In addition, T. asperellum RMCK01 was particularly efficient against Z. morio larvae. On day 21, 88.87%, 88.07%, and 86.73% of insects died when treated with a suspension containing 6 × 108, 3 × 109, and 2.68 × 107 conidia/ml, respectively. The study highlights the potential of this fungal isolate as a biocontrol agent against insect pests.


Background
A major current environmental problem is caused by the indiscriminate use of pesticides under certain agricultural systems (Ortiz-Hernández et al. 2014). Pesticidal spraying can reduce or even eliminate the economic costs caused by plant diseases, insect pests, and weeds. However, there are increasing concerns regarding the overuse Page 2 of 7 Sebumpan et al. Egyptian Journal of Biological Pest Control (2022) 32:47 of pesticides in agriculture as they cause health problems for humans and animals and cause irreversible damage to the ecosystems. Moreover, their use is not always efficient as many pests have developed resistance against pesticides, for instance (Alvarez et al. 2017). Under these circumstances, biological control agents (BCAs) have recovered major importance. The use of beneficial microbes can, for instance, help meet the growing global demand for plant products sustainably, without relying entirely on synthetic pesticides. Members of the fungal genus Trichoderma (Ascomycota: Hypocreaceae) are primarily recognized as BCAs with antagonistic effects against a wide array of fungal phytopathogens, pathogenic bacteria like Ralstonia solanacearum, and lepidopteran insect pests (Mohamed et al. 2020). Trichoderma asperellum (Samuels et al. 1999), although less studied, is also a successful BCA against many plant pathogens (Wu et al. 2017), and its larvicidal potential has been recently recognized (Podder and Ghosh 2019). Some Trichoderma-based products are often commercialized as BCAs against phytopathogens and as biofertilizers or growth promoters (Woo et al. 2014). The biocontrol mechanisms mainly include competition and mycoparasitism, followed by the stimulation of plant resistance and immunity (Sood et al. 2020). Amid their commercial success, a major drawback of these microbial-based fungicides is that not all strains perform the same under different environmental conditions. The collection and characterization of locally adapted strains may increase the availability of highly efficient Trichoderma strains (El Komy et al. 2015).
In this study, a T. asperellum isolate, RMCK01, was isolated, in the southern Philippines (Villaneuva, Misamis Oriental), characterized at the morphological and molecular levels. Its biological control potential was evaluated against the super worm, Zophobas morio (Fabricius, 1776) (Coleoptera: Tenebrionidae) larvae. To our knowledge, this is the first study to assess the biocontrol potential of T. asperellum against a coleopteran insect. The findings of this study pave the way and open the door to the potential application of a new bioinsecticide based on a naturally occurring entomopathogenic fungus.

Fungal isolation
The fungus was isolated from soil samples collected from a dragon fruit (Hylocereus cactus) farm in Villanueva, Misamis Oriental, Philippines (8.59654° N, 124.79971° E), in November 2019. To this end, the insect-baiting technique was used according to the procedure developed by Bedding and Akhurst (1975). Briefly, moistened soil samples were placed in plastic containers. Then, 5 last instar larvae, Z. morio, were added into each container (Navarez et al. 2021). Containers were sealed and stored in the dark at 25 ± 2 °C. Containers were inspected every other day, for 15 days, until visible symptoms of fungal infection were observed. Insect cadavers showing fungal infection were transferred to Sabouraud dextrose agar (SDA-Scharlau) media for purification (Herlinda et al. 2020). To inhibit the growth of bacteria, the media was supplemented with streptomycin, a broad-spectrum antibiotic, at a concentration of 20 µg/l (Goettel and Inglis 1997).

Morphological identification
Isolated fungi were identified based on phenotypic characteristics in SDA and PDA (Potato dextrose agar) media. Characteristics such as surface texture, surface margin, surface topography, surface pigmentation, reverse pigmentation, and growth rate were taken into account. In addition, microscopic characteristics such as the length and shape of conidia were taken into account for initial identification using the key described by Humber (1997).

Molecular identification
Genomic DNA (gDNA) of the fungal isolate was extracted using PureLink ® Genomic Plant DNA Purification Kit (Thermo Fisher Scientific). Gene amplification by PCR was carried out with the following components: 3 µl of gDNA, 1 µl of ITS1 (5′tccgtaggtgaacctgcgg-3′) and 1 µl of ITS4 (5′-tcctccgcttattgatatgc-3′) primers, 2.5 µl of Taq buffer, 0.5 µl of DNA polymerase, 1 µl of dNTP mix, and 1 µl nuclease-free dH 2 O. Cycling parameters on the thermal cycler were set at 95 °C for 5 min, followed by 30 cycles at 95 °C for 60 s, 58 °C for 45 s, 70 °C for 60 s, and a final extension at 72 °C for 10 min. Amplicons generated had sizes ranging from 400 to 600 bp, which was expected for ITS1 and ITS4 primers (Additional file 1: Fig. S1). Capillary sequencing was performed in a bidirectional manner at the Philippine Genome Center-Core Sequencing Facility (Manila, Philippines). Fluorescent-labeled chain terminator dNTPs with the reaction components, viz. amplicons, primers, and ABI BigDye ® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific), were used. Cycling parameters on Bio-Rad T100 Thermal Cycler include: pre-hold at 4 °C; 96 °C for 60 s; 25 cycles of 96 °C for 10 s, 50 °C for 5 s, 62 °C for 4 min and hold at 4 °C. Ethanol precipitation was carried out to remove unincorporated dNTPs, excess primers, and primer dimers. Capillary electrophoresis was carried out on the ABI 3730×l DNA Analyzer using a 50 cm 96-capillary array, POP7TM Polymer, and 3730×l Data Collection Software v3.1. Base-calling was carried out on the Sequencing Analysis Software v5.4.

Sequences alignment and phylogenetic analysis
The generated sequences were manually curated and trimmed in BioEdit (Hall, 1999). The obtained sequences were searched in the National Centre for Biotechnology (NCBI) data bank using the nucleotide basic local alignment search tool (BLASTn) (Altschul et al. 1990). Sequences of Trichoderma asperellum RMCK01 were aligned with sequences of other related fungi species using default ClustalW parameters in MEGA 7.0 (Kumar et al. 2016) and optimized manually in BioEdit (Hall 1999), and sequences were only trimmed at both extremes after alignment to maintain fragments with similar length. Pairwise distances were computed using MEGA 7.0 (Kumar et al. 2016). Codon positions included were 1st + 2nd + 3rd + Non-coding. The base substitution model was evaluated using jModelTest 0.1.1 (Posada 2008). The phylogenetic tree was inferred from the datasets using the Bayesian inference method (BI). All characters were treated as equally weighted and gaps as missing data. Trichoderma longibrachiatum (EU401556.1) and T. brunneoviride (EU518659.1) were used as out-groups. Bayesian phylogenetic reconstructions were performed using MrBayes 3.2.7 (Ronquist et al. 2012). The best fit model was identified as the GTR + G model test using the MrModeltest 2.0 program (Nylander 2004). Metropolis-coupled Markov chains Monte Carlo (MCMCMC) generations were run for 1 × 10 6 cycles, and one tree was retained every 2000 generations. The Bayesian tree was visualized using the FigTree program 1.4.4 (Rambaut 2018). In addition, genetic pairwise distances were estimated using MEGA-7 software (Kumar et al. 2016).

Mass production of spores
Mass production of spores was carried out following the method of Podder and Gosh (2019). For this, 50 g of rice was washed thoroughly under running tap water and soaked in water for 3 h. The soaked rice was transferred to a 15 × 30 cm polypropylene plastic bags and then autoclaved, maintaining a temperature of 121 °C for 15 min under 15 psi. The sterilized rice was allowed to cool down at room temperature. Then, the rice was inoculated by T. asperellum spores under a biological safety cabinet. Trichoderma asperellum spores were produced by growing the fungus on the rice. The inoculated rice was incubated at 28 ± 1 °C for 15 days (Fig. 1A).

Preparation of inoculum and quantification
Conidial concentrations were estimated based on the protocol of Goettel and Inglis (1997), with slight modifications. Briefly, mycelium was harvested, washed with distilled water, and filtered through a cheesecloth. Then, Tween 80 (0.05%) was added and 100 µl of conidial suspension was pipetted and mixed with 100 µl of sterile lactophenol blue stain solution (Barta 2010). This suspension was vortexed for 30 s and serially diluted with sterile deionized water. The number of conidia in each sample was determined using a hemocytometer (Neubauer, Marienfeld, Germany). Then, 3 conidial suspensions were prepared. Spores were counted under microscopic power of 40× and calculated using the formula (Kamaruzzaman et al. 2016): Total spores/ml = Number of spores counted × dilution factor Number of smallest square counted × 4000 Finally, the suspension was standardized to 3 final concentrations using C 1 V 1 = C 2 V 2 formula.

Pathogenicity tests
To test the pathogenicity of T. asperellum isolate RMCK01, the last instar Z. morio larvae were treated with 3 conidial suspensions: 2.68 × 10 7 , 6 × 10 8 , or 3 × 10 9 conidia/ml. These 3 concentrations were selected based on previous studies (Mohamed 2019). Zophobas morio larvae were immersed into one conidial suspension for 60 s and then let to air dry. All insects were then transferred to 9 cm diameter clean glass Petri dishes. A piece of filter paper (9 cm in diameter) was placed at the bottom of the dish with a few drops of water. Controls were dipped into a 0.02% solution of Tween-80 prepared with distilled water. Experiments were repeated 4 times with a new batch of insects and new conidial suspensions, and for each repetition, there were 5 Z. morio larvae. Bioassays were incubated at 26 °C and > 95% relative humidity (RH). Insect mortality and fungal infection symptoms were recorded at 12 h intervals for 21 days (Fig. 1B).

Statistical analysis
Results of statistical analysis are expressed as mean ± standard deviation (n = 3) with 5 replications. To determine whether there were significant differences (p < 0.05), one-way ANOVA was used, followed by Tukey's HSD test (XLSTAT version 2020.1.3) for post hoc analysis.

Morphological characterization
Trichoderma asperellum RMCK01 mycelia were white and dark green, and arranged in 5 concentric rings, each of either white or dark green color when grown for 3 days in PDA media ( Fig. 2A). When grown for 3 days in SDA media, mycelium was white with a hint of green, had a cottony texture, and forms a few concentric rings (Fig. 2B). Conidia were subglobose to globose or ovoidal (Fig. 2C, D) (Table 1).

Molecular characterization and phylogenetic relationships
Trichoderma asperellum RMCK01 had a G + C content of 56.41% and an A + T content of 43.59% in the sequences of ITS rDNA (507 bp) flanked by primers ITS1 and ITS4. These sequences were identical to other ITS rDNA sequences, available from the GenBank, of other T. asperellum strains isolated in Vietnam, India, Thailand, and China, suggesting that they are conspecific. The phylogenetic tree reconstructed using a Bayesian inference model supports this notion (Fig. 3). Trichoderma asperellum RMCK01 formed a well-supported clade with other T. asperellum isolates from Vietnam, India, Thailand, and China. Furthermore, the phylogenetic analyses showed that T. asperellum was a sister group to the clade formed by Trichoderma koningiopsis, T. asperelloides, T. viride, T. yunnanense, and T. hamatum.

Discussion
Use of Trichoderma, as a biocontrol agent against insect pests, has been studied in recent years, uncovering this group of fungi as a promising alternative to be used in  et al. Egyptian Journal of Biological Pest Control (2022) 32:47 sustainable agriculture not only because of its efficiency against plant pathogens but also insect pests (Poveda 2021). The reports of its economic importance in the agricultural industry and the characteristics of Trichoderma that produces large amounts of fungal spores make it ideal for inoculum production under laboratory conditions (Herrera-Téllez et al. 2019). In this study, evidence that T. asperellum naturally occurs in agricultural soils in the Philippines was presented and it can be grown under laboratory conditions and is highly pathogenic against Z. morio larvae. Several studies show the biological control potential of T. asperellum against plant diseases (Vilcacundo et al. 2020); at the same time, it improves plant growth and development (Moreira et al. 2021) in several plant species and also bioremediation qualities are shown (Li et al. 2021). Furthermore, a more recent study reports that this fungal species can have larvicidal effects against Anopheles spp., the vector of malaria and Aedes aegypti (da Silveira et al. 2021). The potential of T. asperellum to control other insect pests has not been shown yet. Hence, to determine the potential of T. asperellum and to control other insect pests, its pathogenicity against Z. morio was evaluated under laboratory conditions. The present experimental results showed that T. asperellum strain RMKC had also a great potential to control Z. morio larvae as the insect mortality was observed a few days after fungal applications, which reached about 86-88% within

Consent for publication
Not applicable. Error bars indicate standard deviations of four replicates with five larvae each. Different letters above error bars indicate significant differences between treatments (Tukey test; p ≤ 0.05)