Evolution of Protein Molecules

Plant pathogens are the main threat for profitable agricultural productivity. Currently, chemical-based pesticides are thought to be an effective and reliable agricultural management measure for controlling pests. Chemical pesticides are highly effective and convenient to use but they are a potential threat for the environment and all kinds of life on earth. Therefore, the use of biological control agents for the management of plant pathogens is considered as a safer and sustainable strategy for safe and profitable agricultural productivity. Bacillus-based biocontrol agents play a fundamental role in the field of biopesticides. Many Bacillus species have proved to be effective against a broad range of plant pathogens. They have been reported as plant growth promoter, systemic resistance inducer, and used for production of a broad range of antimicrobial compounds (lipopeptides, antibiotics and enzymes) and competitors for growth factors (space and nutrients) with other pathogenic microorganisms thr...

https://tandfonline.com/doi/pdf/10.1080/13102818.2017.1286950

Bacillus species as versatile weapons for plant pathogens: a review

Insecticidal activity of Bacillus thuringiensis crystal proteins

Induced plant defenses in response to herbivore attack are modulated by cross-talk between jasmonic acid (JA)- and salicylic acid (SA)-signaling pathways. Oral secretions from some insect herbivores contain effectors that overcome these antiherbivore defenses. Herbivores possess diverse microbes in their digestive systems and these microbial symbionts can modify plant–insect interactions; however, the specific role of herbivore-associated microbes in manipulating plant defenses remains unclear. Here, we demonstrate that Colorado potato beetle (Leptinotarsa decemlineata) larvae exploit bacteria in their oral secretions to suppress antiherbivore defenses in tomato (Solanum lycopersicum). We found that antibiotic-untreated larvae decreased production of JA and JA-responsive antiherbivore defenses, but increased SA accumulation and SA-responsive gene expression. Beetles benefit from down-regulating plant defenses by exhibiting enhanced larval growth. In SA-deficient plants, suppression was not observed, indicating that suppression of JA-regulated defenses depends on the SA-signaling pathway. Applying bacteria isolated from larval oral secretions to wounded plants confirmed that three microbial symbionts belonging to the genera Stenotrophomonas, Pseudomonas, and Enterobacter are responsible for defense suppression. Additionally, reinoculation of these bacteria to antibiotic-treated larvae restored their ability to suppress defenses. Flagellin isolated from Pseudomonas sp. was associated with defense suppression. Our findings show that the herbivore exploits symbiotic bacteria as a decoy to deceive plants into incorrectly perceiving the threat as microbial. By interfering with the normal perception of herbivory, beetles can evade antiherbivore defenses of its host.

https://www.pnas.org/content/pnas/110/39/15728.full.pdf

Herbivore exploits orally secreted bacteria to suppress plant defenses

Bacillus thuringiensis (Bt) was first described by Berliner [10] when he isolated a Bacillus species from the Mediterranean flour moth, Anagasta kuehniella, and named it after the province Thuringia in Germany where the infected moth was found. Although this was the first description under the name B. thuringiensis, it was not the first isolation. In 1901, a Japanese biologist, Ishiwata Shigetane, discovered a previously undescribed bacterium as the causative agent of a disease afflicting silkworms. Bt was originally considered a risk for silkworm rearing but it has become the heart of microbial insect control. The earliest commercial production began in France in, 1938, under the name Sporeine [72]. A resurgence of interest in Bt has been attributed to Edward Steinhaus [105], who obtained a culture in 1942 and attracted attention to the potential of Bt through his subsequent studies. In 1956, T. Angus [3] demonstrated that the crystalline protein inclusions formed in the course of sporulation were responsible for the insecticidal action of Bt. By the early 1980's, Gonzalez et al. [48] revealed that the genes coding for crystal proteins were localized on transmissible plasmids, using a plasmid curing technique, and Schnepf and Whiteley [103] first cloned and characterized the genes coding for crystal proteins that had toxicity to larvae of the tobacco hornworm, from plasmid DNA of Bt subsp. kurstaki HD-1. This first cloning was followed quickly by the cloning of many other cry genes and eventually led to the development of Bt transgenic plants. In the 1980s, several scientists successively demonstrated that plants can be genetically engineered, and finally, Bt cotton reached the market in 1996 [104].

Bacillus thuringiensis as a Specific, Safe, and Effective Tool for Insect Pest Control

Seven closely related xylanolytic, thermophilic bacilli were isolated from mud and water samples from the Gonen and Diyadin hot springs, respectively located in the Turkish provinces of Balikesir and Agri. On the basis of morphology and biochemical characteristics, one of the isolates, designated strain G2T, was studied further. Strain G2T is a xylanolytic, sporulating, Gram-positive, rod-shaped bacterium. The isolate is a thermophilic (optimum temperature for growth, 55–60 °C), facultative anaerobe that grows on a wide range of carbon sources, including glucose, starch, xylose and mannitol. It expressed a high level of xylose isomerase activity on xylose and also on glucose. 16S rRNA gene sequence analysis showed that this isolate resembled Anoxybacillus flavithermus DSM 2641T (>97 % similarity), but 16S–23S rDNA internally transcribed spacer polymorphism PCR showed variation between DSM 2641T and isolate G2T. However, it is also known that analysis of 16S rRNA gene sequences may be insufficient to distinguish between some species. In DNA–DNA hybridization, thermophilic isolate G2T showed relatedness of 53·4 % to A. flavithermus and about 45·0 % to Anoxybacillus pushchinoensis, indicating that it is distinct at the species level. On the basis of the evidence presented, it is proposed that strain G2T (=NCIMB 13933T=NCCB 100040T) be designated as the type strain of Anoxybacillus gonensis sp. nov.

Anoxybacillus gonensis sp. nov., a moderately thermophilic, xylose-utilizing, endospore-forming bacterium

Although Turkey is the first among all hazelnut-producing countries, yield per unit area of this crop is low in comparison to other countries, mainly because many insect species seriously damage hazelnut trees and their fruit. To find effective and safe biocontrol agents, we conducted a survey study to isolate entomopathogenic fungi from the hazelnut-growing region of Turkey and characterized the isolated strains in detail. In addition, we determined the effectiveness of seven selected strains from this region against Melolontha melolontha (Coleoptera: Scarabaeidae) which is one of the most serious pests of hazelnut. In 2006 and 2007, 301 soil samples were collected randomly and analyzed for presence of entomopathogenic fungi using the Galleria bait method. Entomopathogenic fungi were found to occur in 20.59% of the soil samples studied. Based on morphology, ITS sequence and partial sequencing of the 18S (SSU rDNA) and EF1-α genes, the isolates were identified as Metarhizium anisopliae var. anisopliae, Metarhizium sp., Beauveria bassiana, Beauveria cf. bassiana, Isaria fumosorosea and Evlachovaea sp. Metarhizium anisopliae var. anisopliae was isolated from 34 sites and was the most frequent and abundant entomopathogenic species recovered. All the isolates tested were pathogenic to M. melolontha. M. anisopliae var. anisopliae KTU-27 and Evlachovaea sp. KTU-36 produced the highest insecticidal activity (86.6%) within 15 days after inoculation. Our results suggest that entomopathogenic fungi could be good biocontrol agents against M. melolontha, and are discussed with respect to ecology of fungi in relation to habitat in order to evaluate biocontrol potential of these isolates. This is the first study of the distribution of entomopathogenic fungi in the hazelnut-growing region of Turkey and of their pathogenicities against M. melolontha.

Isolation and characterization of entomopathogenic fungi from hazelnut-growing region of Turkey.

Two thermophilic bacilli were isolated from mud and water samples of the Ayder and Kestanbol hot springs in the provinces of Rize and Canakkale, respectively, in Turkey. Strains AB04T and K4T were sporulating, Gram-positive, rod-shaped bacteria. These isolates were moderately thermophilic (with an optimum temperature for growth of 50-55 degrees C), facultative anaerobes able to grow on a wide range of carbon sources including d-glucose, d-raffinose, d-sucrose, D-xylose, D-fructose, L-arabinose, maltose, D-mannose and D-mannitol. Analysis of the 16S rRNA gene sequences showed that these isolates resembled Anoxybacillus flavithermus DSM 2641T and Anoxybacillus gonensis NCIMB 13933T. DNA-DNA hybridization data revealed that thermophilic isolate AB04T has only 51.2 % relatedness to A. flavithermus, 45.1 % relatedness to Anoxybacillus pushchinoensis and 68.6 % relatedness to A. gonensis. Thermophilic isolate K4T showed only 60.4 % relatedness to A. flavithermus, 42.9 % relatedness to A. pushchinoensis and 38.5 % relatedness to A. gonensis. On the basis of the DNA-DNA hybridization data, isolates AB04T and K4T are not related to A. flavithermus DSM 2641T, A. pushchinoensis DSM 12423T or A. gonensis NCIMB 13933T at the species level, but show relatedness to one another of 40.5 %. On the basis of the data presented, it is proposed that strains AB04T (= NCIMB 13972T = NCCB 100050T) and K4T (= NCIMB 13971T = NCCB 100051T) be designated as the type strains of Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov., respectively.

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Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov.

Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum, and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a class I and class II hydrophobin, respectively. To examine function, we deleted all three separately, from the M. brunneum strain KTU-60 genome, using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in virulence and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and virulence.

Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence

The pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.) is one of the most harmful pests to pine species in Mediterranean countries including Turkey. Caterpillars of T. pityocampa are not only significantly harmful to forest trees but also responsible for various allergic reactions in humans and animals. In this study, in order to find a more effective and safe biological control agent against T. pityocampa, we investigated fungal pathogens of T. pityocampa in the Black Sea Region of Turkey and tested their pathogenicity on it. Five different fungi were isolated and identified based on their morphological and molecular characteristics including ITS and partial sequence of EF1-[alpha]. Based on these characteristics, four isolates were identified as Beauveria
 bassiana cf. Clade C (Rehner and Buckley in Mycologia 97:84–98, 2005) and one isolate was identified as Beauveria bassiana. Among these isolates, B. bassiana KTU-24, B.
 bassiana cf. Clade C KTU-66 and KTU-67 showed the highest virulence with 100% mortality within 10 days after application. B. bassiana isolate KTU-24 produced the highest mycosis value with %100. Consequently, B. bassiana KTU-24 seems to be good candidate for further investigation as a possible biological control agent against this pest.

Zihni Demirbag

Papers

Anoxybacillus gonensis sp. nov., a moderately thermophilic, xylose-utilizing, endospore-forming bacterium

Isolation and characterization of entomopathogenic fungi from hazelnut-growing region of Turkey.

Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov.

Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence

Molecular Characterization and Virulence of Beauveria spp. from the Pine Processionary Moth, Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae)