Dual inoculation with rhizosphere-promoting bacterium Bacillus cereus and beneficial fungus Peniophora cinerea improves salt stress tolerance and productivity in willow.

Utilization of rhizosphere microorganisms to improve plant growth and salt tolerance has recently attracted widespread attention. The growth and salt tolerance of willows inoculated with Bacillus cereus JYZ-SD2 and Peniophora cinerea XC were studied under different salt stress conditions. The results showed that the chlorophyll content of willow cuttings inoculated with the XC strain increased significantly by 51.27%. After salt stress of willow cuttings inoculated with B. cereus JYZ-SD2 and P. cinerea XC (solely or in combination), the amount of sodium in the roots from the epidermis to the pericycle decreased and the content of sodium in the pericycle was significantly lower than that of the uninoculated willow, while the proportion of potassium increased. Willow cuttings inoculated with microorganisms showed increased activity of SOD and POD. At the salt concentration of 100 mmol/L, the highest SOD activity was found in B. cereus JYZ-SD2-inoculated willows, with 59.88% increase compared to uninoculated willows; the highest POD activity was found in P. cinerea XC and B. cereus JYZ-SD2 co-inoculated willows, with 51.05% increase compared to uninoculated willows. The Na-K-ATPase and Ca-Mg-ATPase activities of inoculated P. cinerea XC willow cuttings were also 59.38% and 60% higher than that of uninoculated willows, respectively. The qPCR analysis showed that the expression of vp2 gene in the microorganism-inoculated willow leaves was always higher than that in willow alone. The expression of vp2 gene in P. cinerea XC-inoculated willow cuttings was 270.81% higher than that in uninoculated willows. Further observation of the ultrastructure of root cells under salt stress revealed that most of the vesicles in the root tip cells of willow were intact and secreted phagocytic vesicles to absorb sodium ions in the cytoplasm. This study shows that the combined beneficial fungi and rhizosphere-promoting bacteria inoculation technology as a practical biotechnological approach to enhance the growth of willows in salt-affected soils.

Azacoccones F-H, new flavipin-derived alkaloids from an endophytic fungus Epicoccum nigrum MK214079.

Three new flavipin-derived alkaloids, azacoccones F-H (1-3), along with six known compounds (4-9) were isolated from the endophytic fungus Epicoccum nigrum MK214079 associated with leaves of Salix sp. The structures of the new compounds were established by analysis of their 1D/2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) data. The absolute configuration of azacoccones F-H (1-3) was determined by comparison of experimental electronic circular dichroism (ECD) data with reported ones and biogenetic considerations. Epicocconigrone A (4), epipyrone A (5), and epicoccolide B (6) exhibited moderate antibacterial activity against Staphylococcus aureus ATCC 29213 with minimal inhibitory concentration (MIC) values ranging from 25 to 50 µM. Furthermore, epipyrone A (5) and epicoccamide A (7) displayed mild antifungal activity against Ustilago maydis AB33 with MIC values of 1.6 and 1.8 mM, respectively. Epicorazine A (8) showed pronounced cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 1.3 µM.

Copper stress in flooded soil: Impact on enzyme activities, microbial community composition and diversity in the rhizosphere of Salix integra.

Climate change has increased flooding frequency, making the heavy metal polluted areas more vulnerable, and led to increased global land degradation. Information about the alteration of soil microbiota under heavy metal pollution and flooding is still rather limited. Fast-growing trees are candidates for phytoremediation of heavy metal polluted soils. Therefore, the impact of Cu pollution on microbiota in soil used for cultivating Salix integra Thunb. was investigated with and without flooding for 60 d. Bacterial and fungal communities were accessed via partial 16S rRNA (V3-V4) and internal transcribed spacer (ITS) genes. The activity of invertase, urease and cellulase were markedly decreased by 28.5-59%, 55.0-76.7% and 17.3-34.1%, respectively, with increasing Cu levels. Flooding significantly increased the activity of polyphenol oxidase and peroxidase by 56.3% and 41.4% at the highest Cu level compared to its respective non-flooded condition. High Cu concentration significantly decreased the richness and diversity of the bacterial community, and fungi were more sensitive than bacteria under flooding conditions. Redundancy analysis suggests that Cu, Fe and soil organic matter are the key determinants affecting the composition of microbial communities. Our findings provide new insight into the responses of soil microbes to Cu-contamination and contribute to our understanding of metal toxicity in soil-woody plant systems under flooded conditions.

Agromyces aureus sp. nov., isolated from the rhizosphere of Salix caprea L. grown in a heavy-metal-contaminated soil.

A Gram-reaction-positive, motile, yellow-pigmented and rod-shaped bacterial strain, designated AR33T, was isolated from the rhizosphere of Salix caprea L. growing in a former zinc/lead mining and processing site in Austria. A polyphasic approach was applied to determine its taxonomic position. 16S rRNA gene sequence analysis, and morphological and chemotaxonomic properties showed that strain AR33T belongs to the genus Agromyces. Strain AR33T had peptidoglycan type B2γ and the major menaquinones were MK-11, MK-10 and MK-12. The main branched-chain fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Strain AR33T showed catalase and oxidase activity and multiple heavy metal resistances to zinc, lead and cadmium. The DNA G+C content was 70.1 mol%. Levels of 16S rRNA gene sequence similarity with closely related recognized species of the genus Agromyces ranged between 98 and 99 %. However, DNA-DNA hybridization between strain AR33T and the type strains of three Agromyces species showed values lower than 42 % relatedness. Therefore, differential phenotypic characteristics together with DNA-DNA relatedness suggested that strain AR33T can be recognized as representing a distinct Agromyces species, for which the name Agromyces aureus sp. nov. is proposed. The type strain is AR33T (=DSM 101731T=LMG 29235T).

Characterization of cadmium-resistant endophytic fungi from Salix variegata Franch. in Three Gorges Reservoir Region, China.

The community and Cd-resistance of endophytic fungi from roots of Salix variegata Franch. collected from the water-level-fluctuation zone of Three Gorges Reservoir Region, China, were investigated. A total of 53 strains were isolated and identified to 13 morphotaxa, in which Chromosporium, Fusarium and Gonatobotrys were dominant genera. Among them, 27 isolates were selected to measure their resistance to 0.02 mg ml(-1) Cd(2+) and 11 were growth stimulated (Tolerance index>100%). Of these active isolates, four dark septate endophyte (DSE) isolates (Paraphaeosphaeria sp. SR46, Pyrenochaeta sp. SR35, Rhizopycnis vagum SR37 and R. vagum SR44) were further tested for minimum inhibitory concentrations (MICs) against Cd and SR46 was found to be the most tolerant isolate with MIC of 0.39 mg ml(-1). Additionally, the maximum uptake values of these DSEs ranged from 3.01 to 7.89 mg g(-1), but there was no significant correlation between metal uptake with fungal biomass and metal tolerance. Subsequently, a pot experiment was conducted for investigating the impact of SR46 on corn seedlings in Cd-enriched soil. The results obtained suggested that SR46 reduced the Cd bioaccumulation of plant under low (100 mg kg(-1)) Cd stress and enhanced the Cd translocation from root zone to aerial parts under high (200 mg kg(-1)) Cd stress. Besides, it promoted plant growth without Cd stress. These findings indicated S. variegata harbors an endophytic fungal flora showing a high genetic diversity as well as a high level of metal resistance to Cd that has potential values in cadmium cycling and restoration of plant, soil and water system.

Plants' healthiness assessment as part of the environmental monitoring of protected mountainous area in the example of Karkonosze (Giant) Mts. (SW Poland).

The aim of phytopathological monitoring is to check the healthiness of plants and observe the changes that occur in their populations. In the vast majority, these types of observations are conducted in agriculture and forestry. An interesting aspect of phytopathological monitoring is the assessment of the origin of the plant species. The research of fungal communities (including pathogens) in plants may, for example, indicate the relic nature of the plant species. Reduction of the occurrence or disappearance of fungi species associated with its host plant can evidence slow decline of their habitats. This applies mainly to arctic-alpine fungal species. On the other hand, for some plant hosts, colonization of their organs by polyphagous fungi is being recorded. One such example is the downy willow, on which six species of fungi were found in the Karkonosze Mts. In 2014, there were no fungi found on this plant. However, comparing the species composition of fungi associated with downy willow given by Schroeter (1908) to the contemporary one and to the study results of other researchers, a decrease in the number of fungi species is clearly visible. This may be related to the environmental pollution, which took place in the Sudetes in the second half of the twentieth century. For instance, the species of the genus Rhytisma colonize the leaves of trees and shrubs and are particularly sensitive to the concentration of SO2 in the air, but nobody has looked for this fungus on this host in the past. Yet, presently, we were able to find Rhytisma fungus in Karkonosze Mts. Phytopathological monitoring was conducted in the years of 2014-2015 in the sub-alpine zone of the Karkonosze (Giant) Mts. It has been shown that, compared to similar studies conducted in the 1990s of the twentieth century, the species composition of fungi infesting Rubus chamaemorus and Betula carpactica has changed. Is this the beginning of changes that will occur in populations of plants?

Spirosoma endophyticum sp. nov., isolated from Zn- and Cd-accumulating Salix caprea.

A Gram-reaction-negative, yellow-pigmented strain, designated EX36(T), was characterized using a polyphasic approach comprising phylogenetic, morphological and genotypic analyses. The endophytic strain was isolated from Zn/Cd-accumulating Salix caprea in Arnoldstein, Austria. Analysis of the 16S rRNA gene demonstrated that the novel strain is most closely related to members of the genus Spirosoma (95 % sequence similarity with Spirosoma linguale). The genomic DNA G+C content was 47.2 mol%. The predominant quinone was and the major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), C16 : 1ω5c, iso-C17 : 0 3-OH and iso-C15 : 0. On the basis of its phenotypic and genotypic properties, strain EX36(T) should be classified as a novel species of the genus Spirosoma, for which the name Spirosoma endophyticum sp. nov. is proposed. The type strain is EX36(T) ( = DSM 26130(T) = LMG 27272(T)).

Willow volatiles influence growth, development, and secondary metabolism in Aspergillus parasiticus.

Aflatoxin is a mycotoxin and the most potent naturally occurring carcinogen in many animals. Aflatoxin contamination of food and feed crops causes a significant global burden on human and animal health. However, available methods to eliminate aflatoxin from food and feed are not fully effective. Our goal is to discover novel, efficient, and practical methods to control aflatoxin contamination in crops during storage. In the present study, we tested the effect of volatiles produced by willow (Salix acutifolia and Salix babylonica) and maple (Acer saccharinum) bark on fungal growth, development, and aflatoxin production by the fungus Aspergillus parasiticus, one economically important aflatoxin producer. S. acutifolia bark volatiles nearly eliminated aflatoxin accumulation (>90% reduction) by A. parasiticus grown on a minimal agar medium. The decrease in aflatoxin accumulation correlated with a twofold reduction in ver-1 (encodes a middle aflatoxin pathway enzyme) transcript level. Expression data also indicate that one histone H4 acetyltransferase, MYST3, may play a role in epigenetic control of aflatoxin gene transcription in response to volatile exposure. Volatiles derived from wood bark samples also increased fungal growth up to 20% and/or enhanced conidiospore development. Solid-phase microextraction-gas chromatographic-mass spectrometric analysis of bark samples identified sets of shared and unique volatile compounds that may mediate the observed regulatory effects on growth, development, and aflatoxin synthesis. This work provides an experimental basis for the use of willow industry by-products to control aflatoxin contamination in food and feed crops.

Anaerobic ethylene glycol degradation by microorganisms in poplar and willow rhizospheres.

Although aerobic degradation of ethylene glycol is well documented, only anaerobic biodegradation via methanogenesis or fermentation has been clearly shown. Enhanced ethylene glycol degradation has been demonstrated by microorganisms in the rhizosphere of shallow-rooted plants such as alfalfa and grasses where conditions may be aerobic, but has not been demonstrated in the deeper rhizosphere of poplar or willow trees where conditions are more likely to be anaerobic. This study evaluated ethylene glycol degradation under nitrate-, and sulphate-reducing conditions by microorganisms from the rhizosphere of poplar and willow trees planted in the path of a groundwater plume containing up to 1.9 mol l(-1) (120 g l(-1)) ethylene glycol and, the effect of fertilizer addition when nitrate or sulphate was provided as a terminal electron acceptor (TEA). Microorganisms in these rhizosphere soils degraded ethylene glycol using nitrate or sulphate as TEAs at close to the theoretical stoichiometric amounts required for mineralization. Although the added nitrate or sulphate was primarily used as TEA, TEAs naturally present in the soil or CO(2) produced from ethylene glycol degradation were also used, demonstrating multiple TEA usage. Anaerobic degradation produced acetaldehyde, less acetic acid, and more ethanol than under aerobic conditions. Although aerobic degradation rates were faster, close to 100% disappearance was eventually achieved anaerobically. Degradation rates under nitrate-reducing conditions were enhanced upon fertilizer addition to achieve rates similar to aerobic degradation with up to 19.3 mmol (1.20 g) of ethylene glycol degradation l(-1) day(-1) in poplar soils. This is the first study to demonstrate that microorganisms in the rhizosphere of deep rooted trees like willow and poplar can anaerobically degrade ethylene glycol. Since anaerobic biodegradation may significantly contribute to the phytoremediation of ethylene glycol in the deeper subsurface, the need for "pump and treat" or an aerobic treatment would be eliminated, hence reducing the cost of treatment.

ASSOCIATED BACTERIA INCREASE THE PHYTOEXTRACTION OF CADMIUM AND ZINC FROM A METAL-CONTAMINATED SOIL BY MYCORRHIZAL WILLOWS.

In order to enhance phytoremediation efficiency, we investigated the effects of dual inoculation with ectomycorrhizal fungi and the ectomycorrhiza associated bacteria Micrococcus luteus and Sphingomonas sp. on the growth and metal accumulation of willows (Salix viminalis x caprea) on contaminated soil. The bacterial strains were previously collected from sporocarps of ectomycorrhizal fungi. The bacteria increased plant growth and the mycorrhizal dependency of willows colonized with the ectomycorrhizal fungus Hebeloma crustuliniforme. The total cadmium (Cd) and zinc (Zn) accumulation in the shoot biomass was increased after inoculation with the fungal strain Hebeloma crustuliniforme in combination with Micrococcus luteus up to 53% and in combination with Sphingomonas sp. up to 62%, respectively. The dual inoculation in combination with Laccaria laccata did not increase the accumulation of Cd and Zn in the willows. We conclude that associated bacteria can enhance the ectomyorrhiza formation and growth of willows and, thereby, the Cd and Zn accumulation in the plant biomass. The results suggest that bacterial support of root growth promoting ectomycorrhizal fungi may be a promising approach to improve the remediation of metal-contaminated soils by using willows.

Conidial fungi from Salix herbacea leaf litter and their growth temperature preferences

A preliminary qualitative analysis was carried out on fungal composition of leaf litter of Salix herbacea growing on Gran Sasso, Monti della Laga (TE), the highest mountain of the Apennines. Direct isolation in humid chamber and culture of soil on plates were used as methods. The most isolated genera were Acremonium, Cladosporium, Lecanicillium, Mucor, Penicillium and Pestalotia. Lag period and radial growth of the colonies were recorded at different temperatures from -1 to 45ºC. and the optimal temperature for growth and sporulation was detected for each isolate. Most of the isolated strains were psychrotolerant mesophyle and capable to grow within a 1ºC to +30ºC range, except for a thermotolerant strain of Penicillium that could grow at 45ºC .They exhibited a wide range of thermal tolerance together with their ability to grow in habitats which are commonly submitted to great changes in temperature similar to those occurring in alpine climate. Data presented in this paper can be useful to understand the ecological requirements of the fungal components of high mountain ecosystems that are often subject to monitoring in the investigation of the effects of global climatic changes.

Se realizó un análisis cualitativo de la composición fúngica de hojas de la litera de Salix herbacea que crece en Gran Sasso, Monti della Laga(TE), en la altamontaña de los Apeninos. El método utilizado fue el aislamiento directo en cámara humeda y el cultivo del sueloen placas. Los géneros más aislados fueron: Acremonium, Cladosporium, Lecanicillium, Mucor, Penicillium yPestalotia. Se registró la fase lag y el crecimiento radial de las colonias a diferentes temperaturas, desde-1 hasta45°C y se detectó la temperatura óptima de crecimiento y esporulación para cada aislado. La mayoría de las cepas aisladas fueron mesó.las sicrotolerantes y capaces de crecer en un rango de 1°C a +30°C, excepto una cepa de termotolerante de Penicillium que pudo crecer hasta 45°C. Ellas presentaron un amplio rango de tolerancia térmica, destacandose su especialidad para crecer en los hábitats caracterizados por altos cambios de temperatura semejante a los del clima alpino. Los datos presentados en este trabajo pueden ser usados para entender los requerimientosecológicos de los componentes fúngicos de los ecosistemas de alta montaña sujetos a monitoreo en las investigaciones de los efectos del cambio climático global.

Arbuscular mycorrhizal distribution in relation to microsites on recent volcanic substrates of Mt. Koma, Hokkaido, Japan.

Mycorrhizae occur in most terrestrial ecosystems and are crucial to understanding community structure and function. However, their role in primary succession is poorly understood. This study examined the mycorrhizal colonization of six plant species in relation to microsite types on recent volcanic substrates on the summit of Mt. Koma, Hokkaido, Japan. The six microsites were flat, rill, near rock, Carextussock, Polygonum patch and Salix patch. Carex oxyandra was nonmycorrhizal and Agrostis scabra and Campanula lasiocarpa were arbuscular mycorrhizal (AM) at all microsites examined. Agrostis AM colonization levels did not differ across microsites. Near rock Campanula roots contained significantly more hyphae than at flat and Polygonum patch microsites, and rill and Carex tussock Campanula more arbuscules than at Polygonum patches. Penstemon frutescens was found to be facultatively mycotrophic with AM colonization occurring in roots of Penstemon growing in Carex tussocks, Polygonum patches and near rocks. Polygonum weyrichii was found to be ectomycorrhizal. Polygonum located in rills and in Polygonum and Salix patches were more colonized than Polygonum in Carex patches. Salix reinii was heavily ectomycorrhizal.