The Free-Living Stage Growth Conditions of the Endophytic Fungus Serendipita indica May Regulate Its Potential as Plant Growth Promoting Microbe.

Serendipita indica (former Piriformospora indica) is a non-obligate endophytic fungus and generally a plant growth and defence promoter with high potential to be used in agriculture. However, S. indica may switch from biotrophy to saprotrophy losing its plant growth promoting traits. Our aim was to understand if the free-living stage growth conditions (namely C availability) regulate S. indica's phenotype, and its potential as plant-growth-promoting-microbe (PGPM). We grew S. indica in its free-living stage under increasing C availabilities (2-20 g L-1 of glucose or sucrose). We first characterised the effect of C availability during free-living stage growth on fungal phenotype: colonies growth and physiology (plasma membrane proton pumps, stable isotopic signatures, and potential extracellular decomposing enzymes). The effect of the C availability during the free-living stage of the PGPM was evaluated on wheat. We observed that C availability during the free-living stage regulated S. indica's growth, ultrastructure and physiology, resulting in two distinct colony phenotypes: compact and explorer. The compact phenotype developed at low C, used peptone as the major C and N source, and displayed higher decomposing potential for C providing substrates; while the explorer phenotype developed at high C, used glucose and sucrose as major C sources and casein and yeast extract as major N sources, and displayed higher decomposing potential for N and P providing substrates. The C availability, or the C/N ratio, during the free-living stage left a legacy to the symbiosis stage, regulating S. indica's potential to promote plant growth: wheat growth promotion by the explorer phenotype was ± 40% higher than that by the compact phenotype. Our study highlights the importance of considering microbial ecology in designing PGPM/biofertilizers. Further studies are needed to test the phenotypes under more extreme conditions, and to understand if the in vitro acquired characteristics persist under field conditions.

Crop management as a driving force of plant growth promoting rhizobacteria physiology.

Crop management systems influence plant productivity and nutrient use efficiency, as well as plant growth-promoting rhizobacteria (PGPR), which are known to influence the growth of plants via phytohormone production, phosphate solubilization, nitrogen (N) fixation and antimicrobial activity. The objective of this study was to compare the influence of two crop management system on microbial PGPR features. PGPR isolated from the rhizospheres of Carica papaya L. grown under two distinct management systems (conventional and organic) were identified and characterized. The 12 strains most efficient in solubilizing inorganic phosphate belonged to the genera Burkholderia, Klebsiella, and Leclercia. N fixation was observed in the strains B. vietnamiensis from the conventional farming system and B. vietnamiensis, B. cepacia and Leclercia sp. from the organic farming system. The B. vietnamiensis, B. cepacia, Klebsiella sp. and Klebsiella sp. isolates showed antifungal activity, while Leclercia sp. did not. The strains B. vietnamiensis and Enterobcter sp. (isolated from the conventional farming system) and Klebsiella sp. (isolated from the organic farming system) were efficient at solubilizing phosphate, producing phytohormones and siderophores, and inhibiting the mycelial growth of various phytopathogenic fungi (Botrytis cinerea, Pestalotia sp., Alternaria sp., Phoma sp., Fusarium culmorum, Geotrichum candidum). Physiological differences between the isolates from the two crop management regimes were distinguishable after 10 years of distinct management.

Nitrogen-fixing bacteria in Eucalyptus globulus plantations.

Eucalypt cultivation is an important economic activity worldwide. In Portugal, Eucalyptus globulus plantations account for one-third of the total forested area. The nutritional requirements of this crop have been well studied, and nitrogen (N) is one of the most important elements required for vegetal growth. N dynamics in soils are influenced by microorganisms, such as diazotrophic bacteria (DB) that are responsible for biological nitrogen fixation (BNF), so the aim of this study was to evaluate and identity the main groups of DB in E. globulus plantations. Samples of soil and root systems were collected in winter and summer from three different Portuguese regions (Penafiel, Gavião and Odemira). We observed that DB communities were affected by season, N fertilization and moisture. Furthermore Bradyrhizobium and Burkholderia were the most prevalent genera in these three regions. This is the first study describing the dynamic of these bacteria in E. globulus plantations, and these data will likely contribute to a better understanding of the nutritional requirements of eucalypt cultivation and associated organic matter turnover.

Coinfections of Rickettsia slovaca and Rickettsia helvetica with Borrelia lusitaniae in ticks collected in a Safari Park, Portugal.

Borrelia and Rickettsia bacteria are the most important tick-borne agents causing disease in Portugal. Identification and characterization of these circulating agents, mainly in recreational areas, is crucial for the development of preventive measures in response to the gradually increasing exposure of humans to tick vectors. A total of 677 questing ticks including Dermacentor marginatus, Rhipicephalus sanguineus, Ixodes ricinus, Hyalomma lusitanicum, H. marginatum, and Haemaphysalis punctata were collected in a Safari Park in Alentejo, Portugal, to investigate the prevalences of infection and characterize Borrelia and Rickettsia species. From a total of 371 ticks tested by PCR for Borrelia burgdorferi sensu lato (s.l.), of which 247 were tested for Rickettsia, an infection prevalence of 18.3% was found for B. lusitaniae and 55.1% for Rickettsia spp. Sequence analysis of positive amplicons identified the presence of B. lusitaniae (18.3%), R. monacensis strain IRS3 (51.7%), and R. helvetica (48.3%) in I. ricinus. R. slovaca (41.5%), R. raoultii (58.5%), and also B. lusitaniae (21%) were identified in D. marginatus ticks. One (5.9%) H. lusitanicum was infected with B. lusitaniae, and R. massiliae was found in one Rhipicephalus sanguineus. Coinfection was found in 7 (20%) I. ricinus and 34 (23.3%) D. marginatus ticks. We report, for the first time, simultaneous infection with R. helvetica and B. lusitaniae and also R. slovaca, the agent of TIBOLA/DEBONEL, with B. lusitaniae. Additionally, 6 isolates of B. lusitaniae were established, and isolates of Rickettsia were also obtained for the detected species using tick macerates cultured in mammalian and mosquito cell lines. This report describes the detection and isolation of tick-borne agents from a Portuguese Safari Park, highlighting the increased likelihood of infection with multiple agents to potential visitors or staff.

Efficacy of plant extracts against stored-products fungi.

The fungistatic activity of six aqueous extracts of plants were tested against Aspergillus candidus, Aspergillus niger, Penicillium sp. and Fusarium culmorum. The plants were, chamomile (Anthemis nobilis L.), cinnamon (Cinnamomum verum J. Presl.), French lavender (Lavandula stoechas L.), garlic (Allium sativum L.), malva (Malva sylvestris L.) and peppermint (Mentha piperita L.). The more concentrated extracts of chamomile and malva inhibited totally the growth of the tested fungi with malva the most effective one.

Accumulation and depuration of cyanobacterial paralytic shellfish toxins by the freshwater mussel Anodonta cygnea.

The increasing frequency by which the production of paralytic shellfish toxins (PST) by freshwater bloom-forming cyanobacteria is being noticed world-wide raises the possibility of PST bioaccumulation by freshwater mussels. This study evaluates PST accumulation and depuration by the freshwater mussel Anodonta cygnea exposed over a 14-day period to high densities (mean = 1.4 x 10(9) cells1(-1), S.D. = 0.29 x 10(9) cellsl(-1)) of the toxic cyanobacterium Aphanizomenon issatschenkoi (corresponding to a mean toxin concentration of 25.5 nmol PSTl(-1), S.D. = 9.9 nmol PSTl(-1)). Mussels were subsequently detoxified either by starvation or by feeding on the non-toxic green-algae Ankistodesmus falcatus. Filter feeding activity and toxin uptake by the mussels were followed by cell counting and toxin analysis in water samples taken before and after each daily water renewal. The accumulation and depuration of PST as well as the anatomical distribution of toxins were monitored throughout the experiment by HPLC analysis of mussel extracts. Mussels fed the toxic cyanobacterium removed on average 65.3% of cells and 40.36% of total PST daily provided. Daily rates of cell clearance (% of initial) were negatively correlated with the amounts of PST daily provided (but not with the amount of cells). This suggests a negative effect of toxins on the feeding behaviour of mussels. Small amounts of toxins could be detected in the mussels after the second day of exposure, reaching a maximum of 26 microg PST100 g(-1) by day 7. The viscera contained the greatest proportion of toxins (78%) at the start of the toxification. However, increasing amounts of PST were found in the remaining tissues (gills, mantle and foot) over time. Toxins detected in the mussel extracts were the same provided in the dietary A. issatschenkoi. Nevertheless, mussels showed a higher proportion of saxitoxin and decarbomoylsaxitoxin and a lower proportion of gonyautoxin-5 than the fed cyanobacterium. Similar depuration efficiencies were observed among starved individuals (6.9% day(-1)) and those fed with A. falcatus (8.2% day(-1)) indicating that both treatments had comparable effects on toxin metabolism. Mussels showed a typical S shaped depuration kinetics curve consisting of a first short period of slow toxin decay followed by a rapid loss and a subsequent slower release of toxins. Trace to undetectable levels of PST were found in mussels after the 14-day depurating period. Although freshwater mussels are not widely consumed by humans, their capacity to accumulate PST points to the risk of PST propagation through the food chain of freshwater ecosystems via filter-feeding mussels.