Molecular taxonomic characterization and infra-specific diversity of entomopathogenic Beauveria bassiana fungi from Argentina.

It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.

Species Discrimination within the Metarhizium PARB Clade: Ribosomal Intergenic Spacer (rIGS)-Based Diagnostic PCR and Single Marker Taxonomy.

(1) Background: The entomopathogenic fungus Metarhizium anisopliae sensu lato forms a species complex, comprising a tight cluster made up of four species, namely M. anisopliae sensu stricto, M. pinghaense, M. robertsii and M. brunneum. Unambiguous species delineation within this "PARB clade" that enables both the taxonomic assignment of new isolates and the identification of potentially new species is highly solicited. (2) Methods: Species-discriminating primer pairs targeting the ribosomal intergenic spacer (rIGS) sequence were designed and a diagnostic PCR protocol established. A partial rIGS sequence, referred to as rIGS-ID800, was introduced as a molecular taxonomic marker for PARB species delineation. (3) Results: PARB species from a validation strain set not implied in primer design were clearly discriminated using the diagnostic PCR protocol developed. Using rIGS-ID800 as a single sequence taxonomic marker gave rise to a higher resolution and statistically better supported delineation of PARB clade species. (4) Conclusions: Reliable species discrimination within the Metarhizium PARB clade is possible through both sequencing-independent diagnostic PCR and sequencing-dependent single marker comparison, both based on the rIGS marker.

Exploring Bacterial and Fungal Biodiversity in Eight Mediterranean Olive Orchards (Olea europaea L.) in Tunisia.

A wide array of bacteria and fungi are known for their association with pests that impact the health of the olive tree. The latter presents the most economically important cultivation in Tunisia. The microbial diversity associated with olive orchards in Tunisia remains unknown and undetermined. This study investigated microbial diversity to elucidate the microbial interactions that lead to olive disease, and the bio-prospects for potential microbial biocontrol agents associated with insect pests of economic relevance for olive cultivation in the Mediterranean area. Bacterial and fungal isolation was made from soil and olive tree pests. A total of 215 bacterial and fungal strains were randomly isolated from eight different biotopes situated in Sfax (Tunisia), with different management practices. 16S rRNA and ITS gene sequencing were used to identify the microbial community. The majority of the isolated bacteria, Staphylococcus, Bacillus, Alcaligenes, and Providencia, are typical of the olive ecosystem and the most common fungi are Penicillium, Aspergillus, and Cladosporium. The different olive orchards depicted distinct communities, and exhibited dissimilar amounts of bacteria and fungi with distinct ecological functions that could be considered as promising resources in biological control.

COI Haplotyping and Comparative Microbiomics of the Peach Fruit Fly, an Emerging Pest of Egyptian Olive Orchards.

The peach fruit fly, Bactrocera zonata (Tephritidae), is economically relevant as a highly polyphagous pest infesting over 50 host plants including commercial fruit and horticultural crops. As an invasive species, B. zonata was firmly established in Egypt and holds potential to spread further across the Mediterranean basin. The present study demonstrated that the peach fruit fly was found multiplying in olive orchards at two distant locations in Egypt. This is the first report of B. zonata developing in olives. COI barcoding has revealed evidence for high diversity across these peach fruit fly populations. These data are consistent with multiple rather than a single event leading to both peach fruit fly invasion to Egypt and its adaptation to olive. Comparative microbiomics data for B. zonata developing on different host plants were indicative for microbiome dynamics being involved in the adaptation to olive as a new niche with a potential adaptive role for Erwinia or Providencia bacteria. The possibility of symbiont transfer from the olive fruit fly to the peach fruit fly is discussed. Potentially host switch relevant bacterial symbionts might be preferred targets of symbiosis disruption strategies for integrated pest management or biological control of B. zonata.

Bacillus velezensis strain MBY2, a potential agent for the management of crown gall disease.

The reduction of the use chemical pesticides in agriculture is gaining importance as an objective of decision-makers in both politics and economics. Consequently, the development of technically efficient and economically affordable alternatives as, e.g., biological control agents or practices is highly solicited. Crown gall disease of dicotyledonous plants is caused by ubiquitous soil borne pathogenic bacteria of the Agrobacterium tumefaciens species complex, that comprises the species Agrobacterium fabrum and represents a globally relevant plant protection problem. Within the framework of a screening program for bacterial Agrobacterium antagonists a total of 14 strains were isolated from Tunisian soil samples and assayed for antagonistic activity against pathogenic agrobacteria. One particularly promising isolate, termed strain MBY2, was studied more in depth. Using a Multilocus Sequence Analysis (MLSA) approach, the isolate was assigned to the taxonomic species Bacillus velezensis. Strain MBY2 was shown to display antagonistic effects against the pathogenic A. fabrum strain C58 in vitro and to significantly decrease pathogen populations under sterile and non-sterile soil conditions as well as in the rhizosphere of maize and, to a lower extent, tomato plants. Moreover, the ability of B. velezensis MBY2 to reduce C58-induced gall development has been demonstrated in vivo on stems of tomato and almond plants. The present study describes B. velezensis MBY2 as a newly discovered strain holding potential as a biological agent for crown gall disease management.

The pathological effects of Heminecrolysin, a dermonecrotic toxin from Hemiscorpius lepturus scorpion venom are mediated through its lysophospholipase D activity.

We have previously identified Heminecrolysin, a sphingomyelinase D (SMaseD), as the major protein responsible for the main pathological effects observed following Hemiscorpius (H.) lepturus scorpion envenomation. We aimed herein to further investigate the kinetics and molecular mechanisms triggered by Heminecrolysin to initiate hematological disorders and inflammatory reaction. We show that Heminecrolysin highly hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and choline, with a Vmax = 1481 ± 51 µmol/min/mg and a Km = 97 ± 16.78 µM, at a much lesser extend sphingomyelin but not phosphatidylcholine substrates. Its lysophospholipase D (lysoPLD) catalytic efficiency, up to three orders of magnitude higher, comparatively to spider's SMaseDs (newly referred as phospholipases D; PLDs), could explain its strong hemolytic capacity. Chelating agents such as EDTA, EGTA, and 1, 10-phenantroline blocked Heminecrolysin-induced LPC hydrolysis at 98, 48, and 70% respectively. Hemolysis blockade occurs only when the toxin is added to erythrocytes in the presence of serum, source of LPC and complement, indicating that the production of LPA and the presence of complement are mandatory for hemolysis. Moreover, we show that Heminecrolysin efficiently binds to erythrocyte's membrane and provokes phosphatidylserine (PS) translocation without cleavage of glycophorin A, suggesting that, unlike spider's PLDs, complement was activated only via the classical pathway. Interestingly, Heminecrolysin was found to induce PS exposure on human nucleated Jurkat T cells, to stimulate secretion of the pro-inflammatory (TNF-α, IL-6), and anti-inflammatory (IL-10) cytokines by human monocytes, and to provoke a disseminated intravascular coagulation on chick embryo chorioallantoic membrane model system. Taken together, our results indicate that Heminecrolysin evokes the major characteristic clinical features of H. lepturus envenomation by using mainly its lysoPLD, rather than its SMaseD's, activity.