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      Diversity of Bacillus thuringiensis Strains From Qatar as Shown by Crystal Morphology, δ-Endotoxins and Cry Gene Content

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          Abstract

          Bacillus thuringiensis (Bt) based insecticidal formulations have been recognized as one of the most successful, environmentally safe and sustainable method of controlling insect pests. Research teams worldwide are in search of Bt diversity giving more choices of bio-insecticides and alternatives to address insect resistance. In fact, there are many unexplored ecologies that could harbor novel Bt strains. This study is the first initiative to explore Bt strain diversity in Qatar. A collection of 700 Bt isolates was constructed. Scanning electron microscopy of Bt crystals showed different crystal forms, with a high abundance of spherical crystals compared to the bipyramidal ones. Among the spherical crystals, four different morphologies were observed. The δ-endotoxin content of parasporal crystals from each Bt isolate revealed that there are 16 different protein profiles among the isolates of the collection. On the other hand, plasmid pattern analysis showed seven different plasmid profiles. Their insecticidal activity was predicted by exploring the δ-endotoxin coding genes and conducting qualitative insecticidal bioassays. 19 smooth spherical crystal producing isolates have been identified that could be possible candidates for endotoxin production targeting Dipteran insects. Another group of 259 isolates producing bipyramidal and cuboidal crystals could target Lepidopteran and Coleopteran insects. The remaining 422 isolates have novel profiles. In conclusion, Qatari soil ecology provides a good collection and diversity of Bt isolates. In addition to strains harboring genes encoding common endotoxins, the majority are different and very promising for the search of novel insecticidal endotoxins.

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          Insect pathogens as biological control agents: Back to the future.

          The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 1years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance. Insect pathogenic viruses are a fruitful source of microbial control agents (MCAs), particularly for the control of lepidopteran pests. Most research is focused on the baculoviruses, important pathogens of some globally important pests for which control has become difficult due to either pesticide resistance or pressure to reduce pesticide residues. Baculoviruses are accepted as safe, readily mass produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining an increased market share. However, the absence of a practical in vitro mass production system, generally higher production costs, limited post application persistence, slow rate of kill and high host specificity currently contribute to restricted use in pest control. Overcoming these limitations are key research areas for which progress could open up use of insect viruses to much larger markets. A small number of entomopathogenic bacteria have been commercially developed for control of insect pests. These include several Bacillus thuringiensis sub-species, Lysinibacillus (Bacillus) sphaericus, Paenibacillus spp. and Serratia entomophila. B. thuringiensis sub-species kurstaki is the most widely used for control of pest insects of crops and forests, and B. thuringiensis sub-species israelensis and L. sphaericus are the primary pathogens used for control of medically important pests including dipteran vectors. These pathogens combine the advantages of chemical pesticides and MCAs: they are fast acting, easy to produce at a relatively low cost, easy to formulate, have a long shelf life and allow delivery using conventional application equipment and systemics (i.e. in transgenic plants). Unlike broad spectrum chemical pesticides, B. thuringiensis toxins are selective and negative environmental impact is very limited. Of the several commercially produced MCAs, B. thuringiensis (Bt) has more than 50% of market share. Extensive research, particularly on the molecular mode of action of Bt toxins, has been conducted over the past two decades. The Bt genes used in insect-resistant transgenic crops belong to the Cry and vegetative insecticidal protein families of toxins. Bt has been highly efficacious in pest management of corn and cotton, drastically reducing the amount of broad spectrum chemical insecticides used while being safe for consumers and non-target organisms. Despite successes, the adoption of Bt crops has not been without controversy. Although there is a lack of scientific evidence regarding their detrimental effects, this controversy has created the widespread perception in some quarters that Bt crops are dangerous for the environment. In addition to discovery of more efficacious isolates and toxins, an increase in the use of Bt products and transgenes will rely on innovations in formulation, better delivery systems and ultimately, wider public acceptance of transgenic plants expressing insect-specific Bt toxins. Fungi are ubiquitous natural entomopathogens that often cause epizootics in host insects and possess many desirable traits that favor their development as MCAs. Presently, commercialized microbial pesticides based on entomopathogenic fungi largely occupy niche markets. A variety of molecular tools and technologies have recently allowed reclassification of numerous species based on phylogeny, as well as matching anamorphs (asexual forms) and teleomorphs (sexual forms) of several entomopathogenic taxa in the Phylum Ascomycota. Although these fungi have been traditionally regarded exclusively as pathogens of arthropods, recent studies have demonstrated that they occupy a great diversity of ecological niches. Entomopathogenic fungi are now known to be plant endophytes, plant disease antagonists, rhizosphere colonizers, and plant growth promoters. These newly understood attributes provide possibilities to use fungi in multiple roles. In addition to arthropod pest control, some fungal species could simultaneously suppress plant pathogens and plant parasitic nematodes as well as promote plant growth. A greater understanding of fungal ecology is needed to define their roles in nature and evaluate their limitations in biological control. More efficient mass production, formulation and delivery systems must be devised to supply an ever increasing market. More testing under field conditions is required to identify effects of biotic and abiotic factors on efficacy and persistence. Lastly, greater attention must be paid to their use within integrated pest management programs; in particular, strategies that incorporate fungi in combination with arthropod predators and parasitoids need to be defined to ensure compatibility and maximize efficacy. Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are potent MCAs. Substantial progress in research and application of EPNs has been made in the past decade. The number of target pests shown to be susceptible to EPNs has continued to increase. Advancements in this regard primarily have been made in soil habitats where EPNs are shielded from environmental extremes, but progress has also been made in use of nematodes in above-ground habitats owing to the development of improved protective formulations. Progress has also resulted from advancements in nematode production technology using both in vivo and in vitro systems; novel application methods such as distribution of infected host cadavers; and nematode strain improvement via enhancement and stabilization of beneficial traits. Innovative research has also yielded insights into the fundamentals of EPN biology including major advances in genomics, nematode-bacterial symbiont interactions, ecological relationships, and foraging behavior. Additional research is needed to leverage these basic findings toward direct improvements in microbial control.
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            Selective Process for Efficient Isolation of Soil Bacillus spp.

            We were able to isolate Bacillus thuringiensis from environmental samples with a background of 10 bacteria per g of soil. Our selection process differed significantly from classical selection methods which permit only the desired organism to grow. In our process, germination of B. thuringiensis spores was selectively inhibited by sodium acetate, while most of the undesired sporeformers germinated. Next, all of the nonsporulated microbes were eliminated by heat treatment at 80 degrees C for 3 min. The surviving spores were then plated on a rich agar medium and allowed to grow until they sporulated. Of random colonies picked from agar, 20 to 96% were crystal-forming Bacillus species. B. thuringiensis and B. sphaericus were routinely selected by this method.
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              Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis.

              The entire 127,923-bp sequence of the toxin-encoding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis is presented and analyzed. In addition to the four known Cry and two known Cyt toxins, a third Cyt-type sequence was found with an additional C-terminal domain previously unseen in such proteins. Many plasmid-encoded genes could be involved in several functions other than toxin production. The most striking of these are several genes potentially affecting host sporulation and germination and a set of genes for the production and export of a peptide antibiotic.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                11 April 2018
                2018
                : 9
                : 708
                Affiliations
                Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University , Doha, Qatar
                Author notes

                Edited by: Marcus A. Horn, Leibniz University of Hanover, Germany

                Reviewed by: M. Oves, King Abdulaziz University, Saudi Arabia; Beatrix Stessl, Veterinärmedizinische Universität Wien, Austria

                *Correspondence: Samir Jaoua samirjaoua@ 123456qu.edu.qa

                This article was submitted to Terrestrial Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2018.00708
                5904277
                29696009
                9d08e852-56a9-4cdb-a96a-73552cbbb970
                Copyright © 2018 Nair, Al-Thani, Al-Thani, Al-Yafei, Ahmed and Jaoua.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 15 December 2017
                : 27 March 2018
                Page count
                Figures: 5, Tables: 2, Equations: 0, References: 47, Pages: 10, Words: 6679
                Funding
                Funded by: Qatar National Research Fund 10.13039/100008982
                Award ID: GSRA2-1-0604-14015
                Award ID: NPRP 09-705-4-025
                Award ID: QUST-CAS-SPR- 2017-31
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                bacillus thuringiensis,crystals,δ-endotoxins profiling,cry genes,plasmids
                Microbiology & Virology
                bacillus thuringiensis, crystals, δ-endotoxins profiling, cry genes, plasmids

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