Cyanotoxin Screening in BACA Culture Collection: Identification of New Cylindrospermopsin Producing Cyanobacteria

Microcystins (MCs), Saxitoxins (STXs), and Cylindrospermopsins (CYNs) are some of the more well-known cyanotoxins. Taking into consideration the impacts of cyanotoxins, many studies have focused on the identification of unknown cyanotoxin(s)-producing strains. This study aimed to screen strains from the Azorean Bank of Algae and Cyanobacteria (BACA) for MCs, STX, and CYN production. A total of 157 strains were searched for mcy, sxt, and cyr producing genes by PCR, toxin identification by ESI-LC-MS/MS, and cyanotoxin-producing strains morphological identification and confirmation by 16S rRNA phylogenetic analysis. Cyanotoxin-producing genes were amplified in 13 strains and four were confirmed as toxin producers by ESI-LC-MS/MS. As expected Aphanizomenon gracile BACA0041 was confirmed as an STX producer, with amplification of genes sxtA, sxtG, sxtH, and sxtI, and Microcystis aeruginosa BACA0148 as an MC-LR producer, with amplification of genes mcyC, mcyD, mcyE, and mcyG. Two nostocalean strains, BACA0025 and BACA0031, were positive for both cyrB and cyrC genes and ESI-LC-MS/MS confirmed CYN production. Although these strains morphologically resemble Sphaerospermopsis, the 16S rRNA phylogenetic analysis reveals that they probably belong to a new genus.

Morphological and Molecular Characterization of Alternaria sect. Ulocladioides/Ulocladium Isolated from Citrus Fruits in Upper Egypt

Citrus is the most important crop in Upper Egypt. 150 infected samples were collected from citrus samples (Navel orange, Tangerine and Lemon) in Upper Egypt, 50 samples from each fruit. Twenty-two isolates representing three species of Alternaria belong to A. sect. Ulocladioides and A. sect. Ulocladium were isolated on dichloran chloramphenicol- peptone agar (DCPA) medium at 27°C. Tangerine samples were more contaminated with Alternaria followed by Navel orange and no Alternaria species appeared from Lemon samples. Based on the Alt a1 the phylogenetic analysis identified the isolates as Alternaria atra, Alternaria botrytis and Alternaria oudemansii. The pathogenicity of the isolates was tested by inoculation of healthy navel orange, the resulted data showed that all tested isolates were pathogenic to healthy navel orange with different degrees ranged from 31.5±1 - 20±1 mm and A. oudemansii had a low virulent effect. The mycotoxins ability of tested isolates indicated that about 83% of the isolates were TeA toxin producers with amount ranged from 1.54 - 18.47 ug/ml.

Droplet printing reveals the importance of micron-scale structure for bacterial ecology

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology, including whether strains can coexist, which are ecologically dominant, and how productive they are as a community1,2. However, a test of the importance of spatial structure requires manipulation at the fine scales at which this structure naturally occurs3–8. Here we develop a droplet-based printing method to arrange different bacterial genotypes across a sub-millimetre array. We use this to test the importance of fine-scale spatial structure by printing strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins9,10. This reveals that the spatial arrangement of bacterial genotypes is important for ecological outcomes. Toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge such that susceptible strains can coexist with toxin producers, even to the extent that a susceptible strain outnumbers the toxin producer. Head-to-head competitions between toxin producers also reveals strong effects, where spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between two competing strains to understand why space is so important. This reveals the importance of processes that limit the free diffusion of molecules. Specifically, we show that cells closest to a toxin producer bind to and capture toxin molecules, which creates a refuge for their clonemates. Our work provides a new method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in their ecology.

Costs and benefits of provocation in bacterial warfare

Competition in animals involves a wide variety of aggressive behaviors. One of the most sophisticated strategies for a focal actor is to provoke a competitor into uncontrolled aggression toward other competitors. Like animals, bacteria rely on a broad spectrum of molecular weapons, some of which provoke potential rivals by triggering retaliation. While bacterial provocation is well documented, its potential adaptive value has received little attention. Here, we examine the costs and benefits of provocation using mathematical modeling and experiments with Escherichia coli strains encoding colicin toxins. We show that provocation is typically costly in one-to-one encounters because a provoking strain receives a strong reciprocal attack compared with nonprovoking strains. By contrast, provocation can be strongly beneficial in communities including more than two toxin-producing strains, especially when the provoker is shielded from, or resistant to, its opponents’ toxins. In these scenarios, we demonstrate that the benefit of provocation derives from a “divide-and-conquer” effect by which aggression-provoking toxin producers force their competitors into increased reciprocal aggression, leading to their cross-elimination. Furthermore, we show that this effect can be mimicked by using antibiotics that promote warfare among strains in a bacterial community, highlighting the potential of provocation as an antimicrobial approach.

Molecular Characterization of Natural Fungal Flora in Black Olives: From Field to Table

In this study, molecular markers were used to determine fungal flora in black olive fruits from field surveys to the table, following the fermentation process. Field samples were collected from different locations of Canakkale province, including Gokceada (Imbros), where organic farming is employed. Some of the fruits from field samples were used for black table olive production and then fungal flora was tracked during the fermentation process. Fungal isolation was also conducted on some commercial samples. Fifty seven isolates from field samples, 56 isolates from the fermentation process and 17 isolates from commercial products were obtained. Among these isolates, 41 Alternaria, 43 Penicillium, 19 Aspergillus, 8 Monascus and 19 other genera were determined using amplified sizes of the Beta-tubulin gene region. Species level identification was carried out based on sequences of Beta-tubulin amplicons, which provided accurate identification, especially where the genera were morphologically highly similar. The occurrence and prevalence of fungal species changed in fungal collections from the field to the fermentation process. While Alternaria alternata was common in field samples, they were absent during fermentation. Many of these identified species, such as Penicillium expansum, Aspergillus niger and Monascus pilosus, which are known as potential toxin producers such as aflatoxin, ochratoxin A and citrinin, were found both in natural and fermented samples, even at the end of the fermentation process. These results showed that some fungal species which survive on olives from the field to the table are potential toxin producers and can be successfully characterized by amplification and sequencing of Beta-tubulin gene.

Cyanobacteria toxins and toxin producers in nine drinking water reservoirs in Taiwan

Cyanobacteria are present in many drinking water reservoirs in the world, and some of them may produce microcystins, anatoxin-a and other natural toxins: In this study, two groups of algal toxins: microcystins and anatoxin-a, and associated toxin producers were investigated. Water samples from nine major drinking water reservoirs and seven associated water purification plants were collected. An HPLC and an LC/MS were employed to detect the concentrations of five microcystins (microcystins-LR, RR, YR, LW, LF) and anatoxin-a. Molecular biotechnology methods were used to sequence Microcystis sp. gene from the cyanobacteria bloom from two of the reservoirs. Monitoring results suggested that microcystins were present in all the drinking water reservoirs studied, and some of them had concentration higher than the WHO guideline of microcystins-LR (1 μg/L). Unlike microcystins, anatoxin-a was only found in four reservoirs, mostly in King-Men island. After comparing the DNA sequence with existing cyanobacteria reference database, Microcystis aeruginosa and other Microcystis sp. were confirmed present in the two reservoirs tested.

An Isolate of Alternaria alternata That Is Pathogenic to Both Tangerines and Rough Lemon and Produces Two Host-Selective Toxins, ACT- and ACR-Toxins

Two different pathotypes of Alternaria alternata cause Alternaria brown spot of tangerines and Alternaria leaf spot of rough lemon. The former produces the host-selective ACT-toxin and the latter produces ACR-toxin. Both pathogens induce similar symptoms on leaves or young fruits of their respective hosts, but the host ranges of these pathogens are distinct and one pathogen can be easily distinguished from another by comparing host ranges. We isolated strain BC3-5-1-OS2A from a leaf spot on rough lemon in Florida, and this isolate is pathogenic on both cv. Iyokan tangor and rough lemon and also produces both ACT-toxin and ACR-toxin. Isolate BC3-5-1-OS2A carries both genomic regions, one of which was known only to be present in ACT-toxin producers and the other was known to exist only in ACR-toxin producers. Each of the genomic regions is present on distinct small chromosomes, one of 1.05 Mb and the other of 2.0 Mb. Alternaria species have no known sexual or parasexual cycle in nature and populations of A. alternata on citrus are clonal. Therefore, the ability to produce both toxins was not likely acquired through meiotic or mitotic recombination. We hypothesize that a dispensable chromosome carrying the gene cluster controlling biosynthesis of one of the host-selective toxins was transferred horizontally and rearranged by duplication or translocation in another isolate of the fungus carrying genes for biosynthesis of the other host-selective toxin.

Biodiversity of killer activity in yeasts isolated from the Brazilian rain forest

The occurrence of killer activity against a panel composed of 22 industrially and (or) medically important yeasts was investigated in 438 yeast and yeast-like cultures belonging to 96 species, isolated from different environments of the Brazilian rain forest. Altogether, 26% of ascomycetes, 56% of basidiomycetes, and 42% of yeast-like cultures exhibited killer activity against at least one of the panel yeasts. More than 15 species never reported before as toxin producers were found, with Pseudozyma antarctica, Trichosporon asteroides, and Geotrichum klebahnii, showing the broader activity spectra. Plasmid curing did not cure the killer phenotypes of Candida maltosa, Debaryomyces hansenii, G. klebahnii, Tr. asteroides, Cryptococcus laurentii, and Ps. antarctica.Key words: yeasts, killer activity, tropical environments.