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Article

Isolation of Clonostachys rosea and Characterizing Its Entomopathogenic Activity against Dubas Bug (Ommatissus lybicus) Nymphs and Adults

by
Salem S. Al-Nabhani
1,*,
Elham Ahmed Kazerooni
2,
Suad Al-Raqmi
1,
Maryam Al-Hashmi
1,
Shah Hussain
2,
Rethinasamy Velazhahan
2 and
Abdullah M. Al-Sadi
2,3,*
1
Directorate General of Agriculture and Livestock Research, Ministry of Agriculture, Fisheries and Water Resources, Muscat P.O. Box 50, Oman
2
Department of Plant Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khoud P.O. Box 34, Muscat 123, Oman
3
College of Agriculture, University of Al Dhaid, Sharjah P.O. Box 27272, United Arab Emirates
*
Authors to whom correspondence should be addressed.
Agriculture 2024, 14(10), 1770; https://doi.org/10.3390/agriculture14101770
Submission received: 19 July 2024 / Revised: 25 September 2024 / Accepted: 1 October 2024 / Published: 7 October 2024
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

:
The Dubas bug is a serious and widespread pest of date palms in several countries in the Middle East. Chemical pesticides are widely used for managing this pest; however, most pesticides fail due to the continuous development of pesticide resistance. The primary goal of this research was to isolate endophytic fungi and test their entomopathogenic activity against Dubas bug nymphs and adults. A total of 27 fungal isolates were obtained and identified using the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region. These strains represent 16 species, belonging to 10 genera of seven different families, Ascomycota with six families and Basidiomycota with a single family, Quambalariaceae. Due to its remarkable biological control ability against insect pests, Clonostachys rosea was further studied for its entomopathogenic activity against Ommatissus lybicus nymphs and adults in comparison to a commercial strain of Beauveria bassiana. The concentration of 1 × 108 conidia/mL of the selected endophytic isolate was used in lab experiments targeting Dubas bug healthy nymphs and adults. After 7 days of C. rosea treatment, the isolate caused significant mortality rates of 85% in the adult insects, while the mortality rates were moderate in the nymphs (33%). The commercial strain of B. bassiana resulted in 100% mortality levels in nymphs and adults of the Dubas bug. Scanning electron microscopy (SEM) of O. lybicus adults treated with C. rosea and B. bassiana showed mycelium growing on the eye, antenna, cuticle, and egg oviposition parts of O. lybicus. Mycelium growth of both fungi was also evident on the nymphs. This study reports for the first time the isolation of C. rosea from the Dubas bug, and its high entomopathogenic activity against an adult population of O. lybicus.

1. Introduction

Losses in crop yield are caused by a variety of biotic and abiotic factors and are a serious problem for the agriculture sector globally. Agricultural productivity is significantly declining due to insect problems. A vast range of plants, including weeds, crops, forest trees, and medicinal plants, can be consumed by insects. Additionally, insects infest food and other items that are kept in bins, godowns, and packages, which results in significant food waste and a decline in food quality [1]. Pests that inflict damage between 5 and 10% are categorized as minor pests, while those that cause more than 10% harm are considered severe pests [2]. Insect pests cause 18–20% of crop production losses globally; the yearly cost of crop loss is estimated to be USD 470 billion [3].
Date palm (Phoenix dactylifera L., 1753) is a valuable agricultural crop grown primarily in tropical and subtropical countries. Date palm fruits are excellent in nutritional and medicinal value, with significant antioxidant, antiproliferative, and antibacterial characteristics [4]. The most prevalent tree in Oman is the date palm, which produces 377,000 million tons of dates annually, ranking Oman among the 10 largest producers of dates in the world [5]. This tree has a significant presence in Omani society, and it is valued for its social, religious, and agricultural values. It is cultivated in different governorates of Oman and occupies half of the cultivated area.
The primary factor contributing to the reduction in date production is the prevalence of several pests that are present everywhere date palm trees are grown, resulting in high yield and economic losses. Date palm plants are often attacked by insects, but the Dubas bug (Ommatissus lybicus de Bergevin, 1930) poses a special threat to date palm trees in the Sultanate. The Dubas bug invasion has caused a 28% reduction in date palm productivity in Oman [6]. Field surveys are challenging and time-consuming since manually detecting these pests in the field involves significant resources and efforts [7,8,9]. The Dubas bug is a hemimetabolous insect with egg, nymph, and adult stages [10]. It has two generations annually, a spring generation and an autumn generation [10,11,12,13,14]. The Dubas bug is a serious pest in date palms in Oman, Iraq, Iran, Pakistan, Egypt, and Africa [15,16]. It affects date palm growth and yield [17,18]. This pest caused approximately 28% losses of date yield in Oman and nearly 50% in Iraq [19,20,21]. This sucking insect feeds on date palm leaves and fruit stalks excreting large amounts of honeydew. The development of sooty mold and accumulation of dust and insects exuviating on the honeydew reduce photosynthesis, resulting in the change in the color of the leaflets from green to light green or yellow-green. When honeydew is produced in abundance, it drops on intercrops and causes a similar type of damage [22]. In Oman, chemical control using Decis 2.5EC (deltamethrin), Trebon 10EW (etofenprox), Matrixine 2.4EC (oxymatrine), Fytomax 0.1PM (azadirachtin), and Sumithion 50EC (fenitrothion) is common in the management of Dubas bug epidemics, but the development of pesticide resistance is a major issue affecting the efficacy of many of these pesticides [6,7,12,18].
Various fungal microorganisms are harmful to numerous insect species and can inhibit their spread, hence controlling their populations [23]. These fungi are commonly referred to as entomopathogenic fungi. There are about a thousand species that infect and parasitize insects. Entomopathogenic fungi are opportunistic pathogens that have evolved throughout time to infect insects, including the ability to bypass host immune system defenses and the development of cuticular enzymes and degrading chemicals [24,25]. They are worldwide and extensively distributed in nature, present in all habitats, and easily developed in large quantities. Recently, the utilization of entomopathogenic microorganisms, particularly entomopathogenic fungi, has drawn significant attention in scientific studies. Entomopathogens have the potential to lessen the selective pressure that leads to the development of pesticide resistance in insect populations [25,26,27]. Unquestionably, one of their other advantages is that they rarely cause resistance phenomena.
Several types of entomopathogenic fungi, including those from the genera Entomophthora, Cordyceps, Clonostachys, Septobasidium, Beauveria, Metarhizium, and Aspergillus, are known to cause diseases in insect pests that affect agriculture and horticultural crops [1,23,25]. Shaw, et al. [28] isolated approximately 40 fungal taxa from six different genera (Verticillium, Hirsutella, Paecilomyces, Beauveria, Metarhizium, and Tolypocladium) and found them to be pathogenic to mites (Acari). Beauveria bassiana and Metarhizium anisopliae have been effectively used against rice plant hoppers [29,30,31]. Coconut fields suffer from Oryctes, which are controlled by a virus and the fungus Metarhizium anisopliae [32,33]. In Oman, the coconut mite Aceria guerreronis is the most destructive pest in coconut trees in Dhofar. Surveys conducted during 2008–2009 revealed the occurrence of a few entomopathogenic fungi including Hirsutella thompsonii, Cordyceps sp., and Simplicillium sp., which showed a good potential to control the mite [34]. Species of Beauveria and Metarhizium have already been isolated and used against locusts in Oman [35]. Beauveria bassiana, B. brongniartii, and M. anisopliae have also been used in managing red date palm weevil infestations in different countries [36,37].
A few studies investigated the biological control of Dubas bugs using entomopathogenic fungi. Trichoderma harzianum and T. viride were isolated from the mobile stage of the Dubas bug adult and nymph; the mortality of these fungi in the form of spore suspension against nymphal instars of Dubas bugs scored approximately 68.5 and 65.8%, respectively, after 72 h in Iraq [38]. In Iran, Zamani, et al. [39] isolated B. bassiana strains from soil and insects and used them for the biological control of the Dubas bug. It is not clear whether entomopathogenic fungi against the Dubas bug exist naturally in Omani date palm fields.
Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams belongs to the family Bionectriaceae (Ascomycota), and is a highly effective mycoparasite and biological control agent to various fungal pathogens, nematodes, and insects [40]. The biocontrol ability of the fungus is due to its cell wall degrading enzymes, secondary metabolites, and enhancing plant immune system [41]. Clonostachys rosea has been used to suppress the growth of various nematodes including Bursaphelenchus xylophilus Steiner and Buhrer 1934, Caenorhabditis elegans Maupas 1900, Haemonchus contortus (Rudolphi 1803) Cobb 1898, Panagrellus redivivus Linnaeus 1767, and Meloidogyne spp. [42]. It can also parasitize several insect pathogens, such as Myzus persicae Sulzer 1776, Rhopalosiphum padi Linnaeus 1758, Thrips tabaci Lindeman 1889, and Varroa destructor Anderson & Trueman 2000 [43]. However, C. rosae has never been used against the Dubas bug.
Sustainable crop production necessitates eco-friendly management. Biopesticides, which are effective in pest management and the production of sustainable agricultural goods, can be used in place of chemical pesticides [44]. The main objective of this study was to examine the presence of Clonostachys rosea in the Dubas bug (O. lybicus) and evaluate its efficacy against the nymph and adult individuals of this insect. Findings from this study will pave the road for developing integrated management strategies for this important pest.

2. Materials and Methods

2.1. Dubas Bug Sampling and Fungal Isolation

Dead/discolored Dubas bug eggs were gathered from 10 villages, located in three Wilayats (administrative districts) in Oman, with 50 eggs collected from each village; the details are given in Table 1. Examining Dubas bug eggs for fungal isolation rather than adults provides several advantages: it targets a more vulnerable stage in the insect’s life cycle and increases the likelihood of successful infection. Moreover, it allows for a more focused, effective, and potentially safer biological control strategy. The collected eggs were plated on 2.5% potato dextrose agar (PDA, Oxoid Ltd., Basingstoke, Hants, UK) with 100 mg L−1 streptomycin to suppress the growth of bacteria. Each Petri dish was plated with four eggs, which were then incubated at 25 °C for two weeks. The developing fungal colonies on Dubus eggs were transferred to new Petri dishes, to obtain pure cultures using the mycelium tip method [44]. The pure cultures of the isolated fungi were preserved in slant, i.e., 3.5 mL PDA media with 13% glycerol at 4 °C until use (Merck KGaA, Darmstadt, Germany).

2.2. Molecular Identification of Isolates

From the pure cultures, genomic DNA was extracted following the manufacturer’s instructions, using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The fungal isolates were identified using the internal transcribed spacer of nuclear ribosomal DNA (ITS1-5.8S-ITS2 = ITS). The ITS1 and ITS2 primers were used to amplify the ITS region [45]. PCR amplification was performed with PuReTaqTM Ready-To-Go PCR beads (GE Healthcare UK Limited, Buckinghamshire, UK), 1.0 µL of each primer (10 mol/L), 22 µL H2O, and 1 µL template DNA. The PCR conditions were optimized with 30 s of initial denaturation at 98 °C, 35 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension for 5 min at 72 °C, following Al-Sadi and Deadman [46]. At Macrogen Inc. (Seoul, South Korea), the PCR products were purified before being sequenced using the same primers. Sequences generated during this study are deposited at GenBank with accession numbers PQ013209-PQ013235.

2.3. Phylogenetic Analysis

The resulting sequences were trimmed using BioEdit v. 7.2.5 [47] and subjected to BLAST testing to determine sequence similarity to GenBank sequences in the NCBI database. Each genus’s sequences were collected from GenBank, and a unique dataset was built for each genus for a phylogenetic analysis. ClustalX v. 2.1 [48] was used to align and assemble the sequences. The maximum likelihood (ML) method of the phylogenetic analysis was tested with RAxML v. 7.2.6 [49], specifying with the GTR + G model with 1000 bootstrap replicates. A bootstrap percentage (BT) of ≥70% was considered significant. Following species delimitation, a concatenated dataset containing all genera was constructed, and consisted of 135 sequences, including the 27 newly generated sequences, and the outgroup taxon Rhizopus arrhizus (CBS112.07). The dataset was aligned using the online tool of MAFFT v. 7 [50], and manually inspected and adjusted in BioEdit v. 7.0.9 [47], and ClustlX v. 2.1 [48]. The resulting alignment file was subjected to ML phylogeny using RAxML v. 7.2.6 [49], with 1000 bootstrap replicates and the GTR + G model of substitution. FigTree v. 1.4.2 was used to visualize phylogenetic trees, while Adobe Illustrator CC2019 (Adobe Inc., San Jose, CA, USA) was used for tree annotation.

2.4. Bioassay Tests

The obtained Clonostachys rosea isolate was tested against O. lybicus nymphs and adults to determine its pathogenicity. Conidial suspensions were made from PDA-grown, 4-week-old cultures. Conidia were removed from the agar surface using a sterile cell scraper, after adding 10 mL of sterile 0.01% Tween 80 (Sigma Aldrich, St. Louis MO, USA). The conidial suspension was poured into a 50 mL Falcon tube after filtering it through sterile cheesecloth. Spore concentration was measured using a hemocytometer and adjusted to 1 × 108 conidia/mL (this concentration was chosen after conducting a pilot study that involved different concentrations). Conidia were inoculated on PDA, and after 24 h of incubation at 25 °C, the conidia that had germinated were counted to determine when the conidia had begun to sprout. If the germ tubes were as wide as the conidia or wider, the conidia were considered germinated.
O. lybicus adults and nymphs were collected for the pathogenicity test. Adults and nymphs were kept in plastic boxes, fed, and allowed to stay at room temperature for a week to acclimatize to the laboratory environment. Ten healthy nymphs and ten healthy adults were sprayed with the conidial suspension at a concentration of 1 × 108 conidia/mL, each with five replicates; placed on a petri dish; and incubated at 25 °C at 60–80% RH under a 14 h photoperiod. The negative control was sprayed with sterile distilled water, while the positive control was sprayed with a commercial product, Beauveria bassiana (Bals. -Criv.) Vuill., 1912, Katyayani organics, India (1 × 108 conidia/mL). The development of symptoms in nymphs and adults was monitored, and a daily count of dead insects was taken. The fungi were isolated and re-identified using morphological, microscopic, and molecular methods. The experiment was conducted three times. Mortality was calculated using Abbott’s formula [51].

2.5. Sample Preparation for Scanning Electron Microscopic (SEM) Inspection

The growth of C. rosea and B. bassiana on the dead nymphs and adults was examined 7 days following treatment. The nymphs and adults were fixed with 2.5% glutaraldehyde and dehydration using a series of ethanol concentrations [52]. The samples were dried using hexamethyldisilazane (2 washes, 10 min), mounted on standard aluminum stubs, and coated by a gold sputter coater. Eventually, the prepared samples were assessed by scanning electron microscopy (SEM, JEOL JSM 4500LV, Tokyo, Japan).

2.6. Statistical Analysis

Every experiment was carried out in triplicate using a fully randomized design with five replications for every treatment. Using SAS (SAS Institute, Inc., Cary, NC, USA), all data were subjected to a one-way analysis of variance (ANOVA), and percentages were compared using Tukey’s Studentized range test (p < 0.05). The data are shown graphically and provided as a percentage ± standard deviation (SD).

3. Results

3.1. Isolation and Molecular Characterization of Fungal Isolates

A total of 27 fungal isolates were obtained from the field-collected O. lybicus eggs. The isolated fungi were identified using the ITS barcode, representing 16 species belonging to 10 genera, and seven different families. Ascomycota was represented by six families, namely Aspergillaceae, Cladosporiaceae, Microascaceae, Nectriaceae, Pleosporaceae, and Saccotheciaceae, and Basidiomycota with Quambalariaceae. The details of the species are given in Table 1. A single phylogenetic tree for all the isolated species with their respective genera is depicted in Figure 1.

3.2. Laboratory Bioassay

The identified endophytic C. rosea isolate exhibited a strong biocontrol activity and was subjected to a lab bioassay on O. lybicus nymphs and adults. According to the bioassay results, the selected isolate C. rosea that was employed exhibited promising biological activity against O. lybicus (Figure 2). The isolate (C. rosea) resulted in significant mortality rates of 85% in the adult insects and 33% in the nymphs after 7 days of treatment (Figure 3). The commercial product (B. bassiana) had the highest mortality rates, which were found to be 100% in the nymphs and adults after 7 days of treatment (Figure 3). On the other hand, the mortality of adult and nymph insects was significantly low in the control with 7 days of treatment (Figure 3).

3.3. Scanning Electron Microscopy of the Entomopathogenic Fungi on O. lybicus

The growths of C. rosea and B. bassiana on the dead O. lybicus adults treated with the conidia of the tested entomopathogenic fungi are shown in Figure 4 and Figure 5. The growth of the fungus C. rosea was indicated by white spores and mycelium, which formed on the dead insects. Scanning electron microscopy (SEM) of O. lybicus adults and nymphs treated with C. rosea depicted mycelium growing on the eye, antenna, cuticle, and egg oviposition parts of O. lybicus (Figure 4). The hyphal growth of B. bassiana declared by SEM is shown in Figure 5. B. bassiana obviously displayed adhesion and penetration structures on the eye, antenna, cuticle, and egg oviposition parts of O. lybicus.

4. Discussion

Insects are susceptible to a variety of diseases, some of which are caused by fungi. Spores on insects’ cuticles germinate and enter the body, killing the insects. If the conditions are favorable, the fungus will sporulate and produce new spores, and start a new life cycle [40]. These spores can be utilized as biological pesticides, due to their high level of infectivity against Dubas bug insects. Several studies have investigated the efficiency of various entomopathogenic fungal isolates against insect pests [53,54,55].
The entomopathogenic fungus C. rosea has been isolated from different plant species, soil types, and dead insects [56]. However, this fungus has never been isolated from the Dubas bug. C. rosea was isolated for the first time from Dubas bugs’ eggs occurring on date palm trees. The entomopathogenic fungus has been used against several insects but never reported as a biocontrol agent against Dubas bugs. The efficacy of C. rosea has been shown with 100% mortality to Myzus persicae and Aphis fabae Scopoli [56]. C. rosea has also been employed against Bemisia tabaci Gennadius 1889, with 23.5% and 50.4% mortality after six days of inoculation in nymphs and adults, respectively [57]. About 82.5% mortality has been observed in Oncometopia tucumana Schröder 1959, followed 14 days post treatment [58]. In this study, it was found that after 7 days following exposure to C. rosea, the fungus resulted in 33% and 85% mortality in nymphs and adults’ population of O. lybicus, respectively. The variation in insect mortality could be attributed to the insect susceptibility, stage of development, host immune response, environmental conditions, and strain of C. rosea [59].
The present study examined the efficacy of C. rosea, against the nymph and adult stages of the Dubas bug (O. lybicus). Our purpose was to obtain an effective candidate for Dubas bug control, which shows a promising mortality rate against the Dubas bug, compared with the commercially available bioformulations. The obtained results demonstrated that O. lybicus was susceptible to C. rosea. After 7 days following exposure to C. rosea, the fungus resulted in 33% and 85% mortality in the nymphs and adult population of O. lybicus, respectively. On the other hand, B. bassiana was more efficacious, resulting in 100% mortality in the nymphs and adults of the Dubas bug insects. This study suggests that Dubas bug adults are more sensitive to C. rosea infection compared to the nymphs. This could be due to the presence of specific interactions between the adults and the fungus, which deserve further investigation in future studies.
The adhesion and penetration structures of C. rosea and B. bassiana on the dead O. lybicus adults and nymphs were depicted (Figure 5) using SEM. On the infected adults, the fungus C. rosea was growing and there were also indications of hyphal penetrations in the insect cuticle, and the other body parts. As revealed by SEM, the fungus C. rosea formed a dense network of dead insects. Additionally, SEM made it possible to observe the fungus hyphae and spores inside the body of infected adults (Figure 5). These findings indicate that the fungus parasitizes the Dubas bug adults and nymphs.
Species in the genus Clonostachys are often distributed in tropical and subtropical regions, occurring as endophytes, soil dwellers, and plant decomposers [60,61]. Clonostachys has become more popular as a multifunctional biocontrol agent because of its ability to inhibit the sporulation of plant pathogenic fungi, colonize senescent and dead tissues, stimulate plant growth, and enhance plant resistance [62,63]. Specifically, C. rosea is significant because it parasitizes insects and plant pathogenic nematodes in addition to being a necrotrophic mycoparasite of various plant pathogenic fungi [61,63].
A previous study reported the efficacy of C. rosea, as an entomopathogenic fungus, against adult stages of the most problematic coleopteran stored product insect pests in Iraq. Results showed that C. rosea isolates might be used as a biological control agent to prevent pest coleopteran insects that are stored in products [54]. It was demonstrated that C. rosea is a novo-entomopathogenic fungus that affects Amritodus atkinsoni and has the potential to be used in mango hopper pest management [61]. Our findings include the first report on the entomopathogenicity of C. rosea on Dubas bug (O.lybicus) adults and nymphs. However, it is highly desirable to further evaluate the efficacy and specificity of the fungus against the Dubas bug.

5. Conclusions

It is concluded from this study that the entomopathogenic fungus Clonostachys rosae, which has remarkable biocontrol efficiency against several insect pests, also has entomopathogenic activity against the date palm pest, the Dubas bug. Future studies should focus on evaluating the potential effect of the entomopathogenic fungus on the different stages of the Dubas bug under different environmental conditions.

Author Contributions

Conceptualization, S.S.A.-N., A.M.A.-S. and R.V.; methodology, S.S.A.-N., S.A.-R. and M.A.-H.; software, S.S.A.-N. and S.H.; validation, E.A.K. and S.H.; formal analysis, S.S.A.-N., E.A.K. and S.H.; investigation, S.S.A.-N.; resources, A.M.A.-S.; data curation, S.S.A.-N.; writing—original draft preparation, S.S.A.-N.; writing—review and editing, S.S.A.-N., E.A.K., R.V., A.M.A.-S., S.A.-R., M.A.-H. and S.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Ministry of Agriculture, Fisheries and Water Resources (MAFWR).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data are presented in this paper or submitted to GenBank.

Acknowledgments

The authors would like to thank MAFWR and Sultan Qaboos University, Department of Plant Sciences, for providing us with a well-equipped platform to undergo our research activities.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Bihal, R.; Al-Khayri, J.M.; Banu, A.N.; Kudesia, N.; Ahmed, F.K.; Sarkar, R.; Arora, A.; Abd-Elsalam, K.A. Entomopathogenic Fungi: An Eco-Friendly Synthesis of Sustainable Nanoparticles and Their Nanopesticide Properties. Microorganisms 2023, 11, 1617. [Google Scholar] [CrossRef] [PubMed]
  2. Dhaliwal, G.; Jindal, V.; Dhawan, A. Insect pest problems and crop losses: Changing trends. Indian J. Ecol. 2010, 37, 1–7. [Google Scholar]
  3. Arora, R. Breeding Insect Resistant Crops for Sustainable Agriculture; Springer: Singapore, 2017. [Google Scholar]
  4. Alwahshi, K.J.; Saeed, E.E.; Sham, A.; Alblooshi, A.A.; Alblooshi, M.M.; El-Tarabily, K.A.; AbuQamar, S.F. Molecular identification and disease management of date palm sudden decline syndrome in the United Arab Emirates. Int. J. Mol. Sci. 2019, 20, 923. [Google Scholar] [CrossRef] [PubMed]
  5. FAO. FAOSTAT. Available online: http://www.fao.org/faostat/en/#data/QC/visualize (accessed on 26 December 2022).
  6. Al-Khatri, S.A.H. Biological, Ecological and Phylogenic Studies of Pseudoligosita Babylonica Viggiani, a Native Egg Parasitoid of Dubas Bug Ommatissus Lybicus de Bergevin, the Major Pest of Date Palm in the Sultanate of Oman. Ph.D. Thesis, University of Reading, Berkshire, UK, 2011. [Google Scholar]
  7. Al-Kindi, K.M.; Kwan, P.; Andrew, N.R.; Welch, M. Modelling spatiotemporal patterns of dubas bug infestations on date palms in northern Oman: A geographical information system case study. Crop Prot. 2017, 93, 113–121. [Google Scholar] [CrossRef]
  8. Al Shidi, R.H.; Kumar, L.; Al-Khatri, S.A.; Albahri, M.M.; Alaufi, M.S. Relationship of date palm tree density to Dubas bug Ommatissus lybicus infestation in Omani orchards. Agriculture 2018, 8, 64. [Google Scholar] [CrossRef]
  9. Khalaf, M.Z.; Khudhair, M.W. Spatial distribution of dubas bug, Ommatissus lybicus (Homoptera: Tropiduchidae) in date palm frond rows. Int. J. Entomol. Res. 2015, 3, 09–13. [Google Scholar]
  10. Resh, V.H.; Cardé, R.T. Encyclopedia of Insects; Academic Press: Cambridge, MA, USA, 2009. [Google Scholar]
  11. Elwan, A.; Al-Tamimi, S. Life cycle of Dubas bug Ommatissus binotatus lybicus de Berg.(Homoptera: Tropiduchidae) in Sultanate of Oman. Egypt. J. Agric. Res 1999, 77, 1547–1553. [Google Scholar] [CrossRef]
  12. Thacker, J.; Al-Mahmooli, I.; Deadman, M. Population dynamics and control of the dubas bug Ommatissus lybicus in the Sultanate of Oman. In Proceedings of the BCPC International Congress: Crop Science and Technology, Scotland, UK, 10–12 November 2003; Volume 1–2, pp. 987–992. [Google Scholar]
  13. Ba-Angood, S.; Al-Ghurabi, A.; Hubaishan, M. Biology and chemical control of the old world bug (Doubas bug) Ommatissus lybicus DeBerg on bate palm trees in the coastal areas of Hadramout governorate, Republic of Yemen. Arab. J. Plant Prot. 2009, 27, 1–9. [Google Scholar]
  14. Payandeh, A.; Kamali, K.; Fathipour, Y. Population structure and seasonal activity of Ommatissus lybicus in Bam Region of Iran (Homoptera: Tropiduchidae). Munis Entomol. Zool. 2010, 5, 726–733. [Google Scholar]
  15. Mokhtar, A.; Al Nabhani, S. Temperature-dependent development of dubas bug, Ommatissus lybicus (Hemiptera: Tropiduchidae), an endemic pest of date palm, Phoenix dactylifera. Eur. J. Entomol. 2010, 107, 681–685. [Google Scholar] [CrossRef]
  16. Hill, D.; Waller, J. Pests and Diseases of Tropical Crops; Longman: London, UK, 1990; pp. 25–406. [Google Scholar]
  17. MAF (Ed.) Dubas Bug Insect; Ministry of Agriculture: Muscat, Oman, 1981; pp. 5–15.
  18. MAF. Dubas Bug, Ommatissus Lybicus (Homoptera: Tropiduchidae); Ministry of Agriculture: Muscat, Oman, 1997; p. 19.
  19. Kranz, J.; Schmutterer, H.; Koch, W. Diseases, pests, and weeds in tropical crops. Soil Sci. 1978, 125, 272. [Google Scholar] [CrossRef]
  20. Khan, N.; Mukhtar, A.; Alam, M. To Asses the Extent of the Economic Damage Caused by Dubas Bug Ommatissus Binotatus on Date Palms with Particular Reference to Yield and Quality of Fruits; Annual Report of Research Work (1980–1981); Ministry of Agriculture and Fisheries: Muscat, Oman, 1983; pp. 170–179.
  21. Gassouma, M.S. Pests of the date palm (Phoenix dactylifera). In Proceedings of the Regional Workshop on Date Palm Development in the GCC Countries of the Arabian Peninsula, Abu Dhabi, United Arab Emirates, 29–31 May 2004; pp. 11–63. [Google Scholar]
  22. Mokhtar, A. Evaluation of the effectiveness of Thuricide HP on Plutella maculipennis larvae in laboratory test. Malar. J. 1983, 165–169. [Google Scholar]
  23. Islam, W.; Adnan, M.; Shabbir, A.; Naveed, H.; Abubakar, Y.S.; Qasim, M.; Tayyab, M.; Noman, A.; Nisar, M.S.; Khan, K.A. Insect-fungal-interactions: A detailed review on entomopathogenic fungi pathogenicity to combat insect pests. Microb. Pathog. 2021, 159, 105122. [Google Scholar] [CrossRef] [PubMed]
  24. Mora, M.A.E.; Castilho, A.M.C.; Fraga, M.E. Classification and infection mechanism of entomopathogenic fungi. Arq. Do Inst. Biológico 2017, 84, e0552015. [Google Scholar] [CrossRef]
  25. Bava, R.; Castagna, F.; Piras, C.; Musolino, V.; Lupia, C.; Palma, E.; Britti, D.; Musella, V. Entomopathogenic fungi for pests and predators control in beekeeping. Vet. Sci. 2022, 9, 95. [Google Scholar] [CrossRef]
  26. Farenhorst, M.; Mouatcho, J.C.; Kikankie, C.K.; Brooke, B.D.; Hunt, R.H.; Thomas, M.B.; Koekemoer, L.L.; Knols, B.G.; Coetzee, M. Fungal infection counters insecticide resistance in African malaria mosquitoes. Proc. Natl. Acad. Sci. USA 2009, 106, 17443–17447. [Google Scholar] [CrossRef]
  27. Farenhorst, M.; Knols, B.G.; Thomas, M.B.; Howard, A.F.; Takken, W.; Rowland, M.; N’Guessan, R. Synergy in efficacy of fungal entomopathogens and permethrin against West African insecticide-resistant Anopheles gambiae mosquitoes. PLoS ONE 2010, 5, e12081. [Google Scholar] [CrossRef] [PubMed]
  28. Shaw, K.E.; Davidson, G.; Clark, S.J.; Ball, B.V.; Pell, J.K.; Chandler, D.; Sunderland, K.D. Laboratory bioassays to assess the pathogenicity of mitosporic fungi to Varroa destructor (Acari: Mesostigmata), an ectoparasitic mite of the honeybee, Apis mellifera. Biol. Control 2002, 24, 266–276. [Google Scholar] [CrossRef]
  29. Venkat Reddy, A.; Sunitha Devi, R.; Dhurua, S.; Reddy, D.V.V. Study on the efficacy of some entomogenous fungi against brown plant hopper, Nilaparvata lugens Stal in irrigated rice. J. Biopestic. 2013, 6, 139–143. [Google Scholar] [CrossRef]
  30. Nagendra Reddy, B.; Jhansi Lakshmi, V.; Uma Maheswari, T.; Laha, G.S. Compatibility of entomopathogenic fungi with neonicotinoids for the management of brown plant hopper of rice, nilaparvata lugens stal. (delphacidae: Hemiptera). Agric. Sci. Dig. 2021, 41, 324–328. [Google Scholar] [CrossRef]
  31. Kumar, S.; Singh, H. Studies on the influence of insecticides and bio-pesticides for the management of Brown plant hopper, Nilaparvata lugens (Stal) in the condition of western U.P. (India). Int. J. Trop. Insect Sci. 2021, 41, 1441–1449. [Google Scholar] [CrossRef]
  32. Gopal, M.; Gupta, A.; Sathiamma, B.; Nair, C.P.R. Microbial pathogens of the coconut pest Oryctes rhinoceros: Influence of weather factors on their infectivity and study of their coincidental ecology in Kerala, India. World J. Microbiol. Biotechnol. 2002, 18, 417–421. [Google Scholar] [CrossRef]
  33. Mani, M.; Krishnamoorthy, A.; Ramanujam, B. Trends in the Biological Control of Horticultural Crop Pests in India. In Trends in Horticultural Entomology; Springer: Singapore, 2022; pp. 243–281. [Google Scholar]
  34. MAF. Developing a mass production technique for Hirsutella thomposonii for control of the coconut mites. In Agriculture and Livestock Research Annual Report; Ministry of Agriculture: Muscat, Oman, 2013; pp. 43–44. [Google Scholar]
  35. Moghal, S.M.; Shivanathan, P.; Mani, A.; Al-Zidjali, A.D.; Al-Zidjali, T.S.; Al-Raeesy, Y.M. Status of Pests and Diseases in Oman: Series 1: Plant Diseases in the Batinah; Ministry of Agriculture and Fisheries: Muscat, Oman, 1993.
  36. Sabbahi, R.; Hock, V. Entomopathogenic fungi against the red palm weevil: Lab and field evidence. Crop Prot. 2024, 177, 106566. [Google Scholar] [CrossRef]
  37. Dembilio, O.; Jacas, J.A. Bio-ecology and integrated management of the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae), in the region of Valencia (Spain). Hell. Plant Prot. J. 2012, 5, 1–12. [Google Scholar]
  38. Sfih, M.S.; Mehid, H.M.R.; Al-Pnayan, M.A. Effect of Some Fungi and Plant Extracts in Ommatissus binotatus var. lybicus De Berg. (Tropiduchidae: Homoptera). Basrah J. Date Palm Res. 2009, 8, 117–133. [Google Scholar]
  39. Zamani, Z.; Aminaee, M.M.; Khaniki, G.B. Introduction of Beauveria bassiana as a biological control agent against the Ommatissus lybicus in Kerman province. Arch. Phytopathol. Plant Prot. 2013, 46, 1821–1830. [Google Scholar] [CrossRef]
  40. Sun, Z.-B.; Li, S.-D.; Ren, Q.; Xu, J.-L.; Lu, X.; Sun, M.-H. Biology and applications of Clonostachys rosea. J. Appl. Microbiol. 2020, 129, 486–495. [Google Scholar] [CrossRef]
  41. Mouekouba, L.D.; Zhang, L.; Guan, X.; Chen, X.; Chen, H.; Zhang, J.; Zhang, J.; Li, J.; Yang, Y.; Wang, A. Analysis of Clonostachys rosea-induced resistance to tomato gray mold disease in tomato leaves. PLoS ONE 2014, 9, e102690. [Google Scholar] [CrossRef]
  42. Rodríguez-Martínez, R.; Mendoza-de-Gives, P.; Aguilar-Marcelino, L.; López-Arellano, M.E.; Gamboa-Angulo, M.; Hanako RosasSaito, G.; Reyes-Estébanez, M.; Guadalupe García-Rubio, V. In vitro lethal activity of the nematophagous fungus Clonostachys rosea (Ascomycota: Hypocreales) against nematodes of five different taxa. BioMed Res. Int. 2018, 2018, 3501827. [Google Scholar] [CrossRef]
  43. Muvea, A.M.; Meyh€ofer, R.; Subramanian, S.; Poehling, H.M.; Ekesi, S.; Maniania, N.K. Colonization of onions by endophytic fungi and their impacts on the biology of Thrips tabaci. PLoS ONE 2014, 9, e108242. [Google Scholar] [CrossRef]
  44. Irsad; Shahid, M.; Haq, E.; Mohamed, A.; Rizvi, P.Q.; Kolanthasamy, E. Entomopathogen-based biopesticides: Insights into unraveling their potential in insect pest management. Front. Microbiol. 2023, 14, 1208237. [Google Scholar] [CrossRef] [PubMed]
  45. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc. A Guide Methods Appl. 1990, 18, 315–322. [Google Scholar]
  46. Al-Sadi, A.M.; Deadman, M.L. Influence of seed-borne Cochliobolus sativus (Anamorph Bipolaris sorokiniana) on crown rot and root rot of barley and wheat. J. Phytopathol. 2010, 158, 683–690. [Google Scholar] [CrossRef]
  47. Hall, T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In Proceedings of the Nucleic Acids Symposium Series, London, UK, 12–14 July 1999; pp. 95–98. [Google Scholar]
  48. Larkin, M.A.; Blackshields, G.; Brown, N.P.; Chenna, R.; McGettigan, P.A.; McWilliam, H.; Valentin, F.; Wallace, I.M.; Wilm, A.; Lopez, R. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23, 2947–2948. [Google Scholar] [CrossRef] [PubMed]
  49. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef]
  50. Katoh, K.; Rozewicki, J.; Yamada, K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2018, 20, 1160–1166. [Google Scholar] [CrossRef]
  51. Abbott, W.S. A method of computing the effectiveness of an insecticide. J. Econ. Entomol 1925, 18, 265–267. [Google Scholar] [CrossRef]
  52. Gabarty, A.; Salem, H.M.; Fouda, M.A.; Abas, A.A.; Ibrahim, A.A. Pathogencity induced by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in Agrotis ipsilon (Hufn.). J. Radiat. Res. Appl. Sci. 2014, 7, 95–100. [Google Scholar] [CrossRef]
  53. Mohammed, A.A.; Younus, A.S.; Ali, A.N. Efficacy of Clonostachys rosea, as a promising entomopathogenic fungus, against coleopteran stored product insect pests under laboratory conditions. Egypt. J. Biol. Pest Control 2021, 31, 55. [Google Scholar] [CrossRef]
  54. Khashaveh, A.; Safaralizadeh, M.H.; Ghosta, Y. Pathogenicity of Iranian isolates of Metarhizium anisopliae (Metschinkoff)(Ascomycota: Hypocreales) against Trogoderma granarium Everts (Coleoptera: Dermestidae). Biharean Biol. 2011, 5, 51–55. [Google Scholar]
  55. Mohammed, A.A.; Kadhim, J.H.; Hasan, A.M. Laboratory evaluation of entomopathogenic fungi for the control of khapra beetle (Coleoptera: Dermestidae) and their effects on the beetles’ fecundity and longevity. J. Agric. Urban Entomol. 2019, 35, 1–11. [Google Scholar] [CrossRef]
  56. Mohammed, A.A.; Ahmed, F.A.; Younus, A.S.; Kareem, A.A.; Salman, A.M. Molecular identification of two entomopathogenic fungus Clonostachys rosea strains and their efficacy against two aphid species in Iraq. J. Genet. Eng. Biotechnol. 2022, 20, 67. [Google Scholar] [CrossRef] [PubMed]
  57. Anwar, W.; Ali, S.; Nawaz, K.; Iftikhar, S.; Javed, M.A.; Hashem, A.; Alqarawi, A.A.; Abd Allah, E.F.; Akhter, A. Entomopathogenic fungus Clonostachys rosea as a biocontrol agent against whitefly (Bemisia tabaci). Biocontrol Sci. Technol. 2018, 28, 750–760. [Google Scholar] [CrossRef]
  58. Toledo, A.; Virla, E.; Humber, R.; Paradell, S.; Lastra, C.L. First record of Clonostachys rosea (Ascomycota: Hypocreales) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimiagrossa (Hemiptera: Cicadellidae) in Argentina. J. Invertebr. Pathol. 2006, 92, 7–10. [Google Scholar] [CrossRef] [PubMed]
  59. Bidochka, M.J.; Khachatourians, G.G. Identification of Beauveria bassiana extracellular protease as virulence factor in pathogenicity towards the migratory grasshopper Melanoplus sanguinipes. J. Invertebr. Pathol. 1990, 56, 362–370. [Google Scholar] [CrossRef]
  60. Schroers, H.-J. A monograph of Bionectria (Ascomycota, Hypocreales, Bionectriaceae) and Its Clonostachys Anamorphs Studies in Mycology; Centraalbureau voor Schimmelcultures (CBS): Utrecht, The Netherlands, 2001; p. 212. [Google Scholar]
  61. Tamta, A.K.; Pandey, R.; Sharma, J.R.; Rai, R.; Barman, M.; Deeksha, M.G.; Mitra, D.; Mohapatra, P.K.D.; Sami, R.; Al-Mushhin, A.A. First Record of Clonostachys rosea (Ascomycota: Hypocreales) Entomopathogenic Fungus in the Mango Hopper Amritodus atkinsoni (Hemiptera: Cicadellidae). Pathogens 2022, 11, 1447. [Google Scholar] [CrossRef]
  62. Nobre, S.A.; Maffia, L.A.; Mizubuti, E.S.; Cota, L.V.; Dias, A.P.S. Selection of Clonostachys rosea isolates from Brazilian ecosystems effective in controlling Botrytis cinerea. Biol. Control 2005, 34, 132–143. [Google Scholar] [CrossRef]
  63. Mascarin, G.M.; da Silva, A.V.R.; da Silva, T.P.; Kobori, N.N.; Morandi, M.A.B.; Bettiol, W. Clonostachys rosea: Production by Submerged Culture and Bioactivity Against Sclerotinia sclerotiorum and Bemisia tabaci. Front. Microbiol. 2022, 13, 851000. [Google Scholar] [CrossRef]
Agriculture 14 01770 g001
Figure 1. The maximum likelihood phylogenetic tree based on ITS sequences; specimens in the red font are the species isolated during this study; above the nodes are the values of ML bootstrap percentages (≥70%) that are considered significant.
Figure 1. The maximum likelihood phylogenetic tree based on ITS sequences; specimens in the red font are the species isolated during this study; above the nodes are the values of ML bootstrap percentages (≥70%) that are considered significant.
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Figure 2. Treated nymphs/adults of Ommatissus lybicus by Beauveria bassiana (A,B), Clonostachys rosea (C,D), and water (E,F) in pathogenicity tests.
Figure 2. Treated nymphs/adults of Ommatissus lybicus by Beauveria bassiana (A,B), Clonostachys rosea (C,D), and water (E,F) in pathogenicity tests.
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Figure 3. Mortality rates of Ommatissus lybicus nymphs (A) and adults (B) treated with Beauveria bassiana and Clonostachys rosea isolates (1 × 108 conidia/mL) and sterile distilled water as a control. Values in the linear graph followed by the same lowercase letters indicate no significant differences between data points on the same day (ANOVA test, p > 0.05).
Figure 3. Mortality rates of Ommatissus lybicus nymphs (A) and adults (B) treated with Beauveria bassiana and Clonostachys rosea isolates (1 × 108 conidia/mL) and sterile distilled water as a control. Values in the linear graph followed by the same lowercase letters indicate no significant differences between data points on the same day (ANOVA test, p > 0.05).
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Figure 4. Mycelial growth and development of Clonostachys rosea on eye (A), antenna (B), cuticle (C), and egg oviposition parts (D) of Ommatissus lybicus (adult) and on eye (E) and mouth parts (F) of Ommatissus lybicus (nymph) assessed by scanning electron microscope (SEM).
Figure 4. Mycelial growth and development of Clonostachys rosea on eye (A), antenna (B), cuticle (C), and egg oviposition parts (D) of Ommatissus lybicus (adult) and on eye (E) and mouth parts (F) of Ommatissus lybicus (nymph) assessed by scanning electron microscope (SEM).
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Figure 5. Mycelial growth and development of Beauveria bassiana on eye (A), mouth (B), cuticle (C), and egg oviposition parts (D) of Ommatissus lybicus (adult) and cuticle (E) and mouthparts (F) of Ommatissus lybicus (nymph) assessed by scanning electron microscope (SEM).
Figure 5. Mycelial growth and development of Beauveria bassiana on eye (A), mouth (B), cuticle (C), and egg oviposition parts (D) of Ommatissus lybicus (adult) and cuticle (E) and mouthparts (F) of Ommatissus lybicus (nymph) assessed by scanning electron microscope (SEM).
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Table 1. Fungal isolates from the infected Dubas eggs in different villages of Oman.
Table 1. Fungal isolates from the infected Dubas eggs in different villages of Oman.
WilayatVillageFungi Isolated from Dubas Egg
Al HamraMesfat alabriynCanariomyces microsporus
Cladosporium sphaerospermum
Cladosporium endophyticum
Cladosporium oxysporum
SamailFalaj almaraghahAlternaria alternata species complex
AlaainhAlternaria alternata species complex
SifalatAureobasidium iranianum
Cladosporium sphaerospermum
Aspergillus sydowii
Quambalaria cyanescens
Xenoacremonium falcatum
AlayateAureobasidium iranianum
Cladosporium sphaerospermum
IzkiQarrot SouthCladosporium colombiae
Al humaydahClonostachys rosea
Cladosporium sphaerospermum (two isolates)
Quambalaria cyanescens
Penicillium sizovae
SaimaAlternaria alternata species complex
Cladosporium sphaerospermum
Fusarium fujikuroi SC
MuqazzahAureobasidium subglaciale
Al qariyatainCladosporium sphaerospermum (two isolates)
Cladosporium halotolerans
Penicillium steckii
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MDPI and ACS Style

Al-Nabhani, S.S.; Kazerooni, E.A.; Al-Raqmi, S.; Al-Hashmi, M.; Hussain, S.; Velazhahan, R.; Al-Sadi, A.M. Isolation of Clonostachys rosea and Characterizing Its Entomopathogenic Activity against Dubas Bug (Ommatissus lybicus) Nymphs and Adults. Agriculture 2024, 14, 1770. https://doi.org/10.3390/agriculture14101770

AMA Style

Al-Nabhani SS, Kazerooni EA, Al-Raqmi S, Al-Hashmi M, Hussain S, Velazhahan R, Al-Sadi AM. Isolation of Clonostachys rosea and Characterizing Its Entomopathogenic Activity against Dubas Bug (Ommatissus lybicus) Nymphs and Adults. Agriculture. 2024; 14(10):1770. https://doi.org/10.3390/agriculture14101770

Chicago/Turabian Style

Al-Nabhani, Salem S., Elham Ahmed Kazerooni, Suad Al-Raqmi, Maryam Al-Hashmi, Shah Hussain, Rethinasamy Velazhahan, and Abdullah M. Al-Sadi. 2024. "Isolation of Clonostachys rosea and Characterizing Its Entomopathogenic Activity against Dubas Bug (Ommatissus lybicus) Nymphs and Adults" Agriculture 14, no. 10: 1770. https://doi.org/10.3390/agriculture14101770

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