Assessment of phylloplane yeasts on selected Mediterranean plants by FISH with group- and species-specific oligonucleotide probes

A previous culture-dependent survey of phylloplane yeasts from selected Mediterranean plants showed that a few species were present in high densities in almost all leaf samples, regardless of the plant type, location or sampling season. However, a few species appeared to be restricted to Cistus albidus leaves, namely Cryptococcus cistialbidi . Here, we describe a culture-independent FISH assay to detect and quantify whole yeast cells in leaf washings. After optimization, the technique was used to check the apparent association between C. albidus leaves and C. cistialbidi and the abundance and ubiquity of other basidiomycetous yeast species such as Erythrobasidium hasegawianum and Sporobolomyces spp. in leaf samples from this and other neighboring plants ( Acer monspessulanum and Quercus faginea ). No yeast cells were detected in Pistacia lentiscus leaf samples. We were also able to demonstrate that three phylloplane yeasts ( C. cistialbidi, E. hasegawianum and Sporobolomyces spp.) appeared to be log-normally distributed among individual C. albidus leaves. The log-normal distribution has important implications for the quantification of phylloplane yeasts based on the washing and plating of bulk leaf samples, which will tend to overestimate the size of the respective populations and become an error source in yeast surveys or related biocontrol studies.     

Phylloplane yeasts from Portugal: seven novel anamorphic species in the Tremellales lineage of the Hymenomycetes (Basidiomycota) producing orange-coloured colonies

A survey of epiphytic yeasts on leaves of selected Mediterranean plant species collected at the 'Arrábida Natural Park' (Portugal) yielded about 850 isolates, mostly of basidiomycetous affinity. Amongst the basidiomycetes, 35 strains showed the following characteristics: production of orange-coloured colonies, ability to produce starch-like compounds, assimilation of D-glucuronic acid and/or inositol, inability to utilize nitrate, and formation of ballistoconidia by many of the isolates. This group of yeasts was assigned to the Tremellales lineage of the Hymenomycetes and was further characterised using a combination of conventional phenotypic identification tests with molecular methods, namely PCR fingerprinting and rDNA sequencing. Eight additional strains presumptively identified as Bullera armeniaca, B. crocea or Cryptococcus hungaricus were also studied. Twenty-eight strains could be assigned to or were phylogenetically related to recognised species of Dioszegia in the 'Luteolus clade', but the 15 remaining strains belonged to other clades within the Tremellales. Ten phylloplane isolates were identified as Dioszegia hungarica, one as D. aurantiaca, another as D. crocea and three others were ascribed to the recently described species D. zsoltii. Seven novel species, viz. Cryptococcus amylolyticus, C. armeniacus, C. cistialbidi, Dioszegia buhagiarii, D. catarinonii, D. fristingensis and D. takashimae, are proposed for the remaining strains that did not correspond to any of the hitherto recognised species.     

Restoration of a Limestone Quarry: Effect of Soil Amendments on the Establishment of Native Mediterranean Sclerophyllous Shrubs

Limestone quarries are spread over the Mediterranean Basin and have a strong environmental impact on the landscape, causing vegetation losses and soil losses. A reclamation project was conducted in a limestone quarry, situated in Arrábida Natural Park (southwest Portugal), that is dominated by mediterranean vegetation. Revegetation was conducted using three evergreen sclerophyllous shrub species (Ceratonia siliqua, Olea europaea, and Pistacia lentiscus), and new techniques were assayed to improve plant water status and nutrient status during the first phases after plantation. A water‐holding polymer (gel), fertilizer, and mycorrhiza inoculum were applied in a factorial experiment in a randomized complete block design. The success of these techniques was evaluated during 1.5 years, through monitoring of growth and ecophysiology of plants. Plant survival was high, the lowest values (95%) being recorded in C. siliqua. There were species‐specific responses to the treatments applied. Ceratonia siliqua showed the highest growth rates and was the only species with growth stimulated by fertilizer application. However, the application of fertilizer induced changes in leaf characteristics of the other two species, increasing chlorophyll and nitrogen contents. Mycorrhiza inoculum had no effect on plant response. The addition of the water‐holding polymer induced higher midday plant water potentials in C. siliqua, O. europaea, and P. lentiscus, but in the latter two species the simultaneous addition of gel and fertilizer induced the lowest water potentials. The addition of fertilizer and gel is recommended in future revegetation programs but not the combination of both when revegetating with O. europaea and P. lentiscus. The results of this experiment indicate that the use of these mediterranean species, adapted to nutrient and water stress, can circumvent harsh conditions of the quarry.     

Coenological research on macrofungi in evergreen oak woods in the hills near Siena (Italy)

For a mycocoenological study of macromycetes in Mediterranean evergreen forests in the Siencese province, five stands belonging to theQuercion ilicis have been observed during more than two years. The phytosociological analysis of these evergreen oak woods, situated at the limit of the geographical distribution of the alliance, reveals them as intermediate between the typical Mediterranean vegetation and the sub-Mediterranean deciduous broad-leaved forests. 181 fungal species were recognized and assigned to seven ecological groups on the basis of substratum, habitat, forest management and/or particular mycorrhizal relationship. Our results have been compared with those obtained in some European deciduous forests. Such a comparison shows the occurrence of 16 thermophilous fungal species in the Siena woods which are missing or very rare in the European deciduous forests. On the basis of our present knowledge, five of these species (Boletus lepidus, Hygrophorus dichrous, Hygrocybe nigrescens, Inocybe similis, Phellinus torulosus) may be considered as strictly tied to the evergreen oak woods. Five others, which have been reported for several European phytocoenoses, seem to be ‘preferential taxa’ of our evergreen oak woods, namely:cortinarius calochrous, C. sodagnitus, Hygrophorus arbustivus, H. russula, Lyophyllum immundum. On the whole the highest similarities have been found with the more xerothermophilous European forests (Querco-Lithospermetum andSorbo-Quercetum). The presence in theQuercus ilex woods of numerous more mesophilous fungi, some of which are widespread in beech forests, underlines the transitional nature of the phytocoenoses studied.     

Culturable Fungi of Stored ‘Golden Delicious’ Apple Fruits: A One-Season Comparison Study of Organic and Integrated Production Systems in Switzerland

The effects of organic and integrated production systems on the culturable fungal microflora of stored apple fruits from five matched pairs of certified organic and integrated ‘Golden Delicious’ farms were studied at five representative production sites in Switzerland. Isolated fungi were identified morphologically. Colonization frequency (percentage of apples colonized), abundance (colony numbers), and diversity (taxon richness) were assessed for each orchard. The standard quality of the stored fruits was comparable for both organic and integrated apples and complied with national food hygiene standards. Yeasts (six taxa) and the yeast-like fungus Aureobasidium pullulans were the dominant epiphytes, filamentous fungi (21 taxa) the dominant endophytes. The most common fungi occurred at all sites and belonged to the “white” and “pink” yeasts, yeast-like A. pullulans, filamentous fungi Cladosporium spp., Alternaria spp., and sterile filamentous fungi. Canonical correspondence analysis of the total fungal community revealed a clear differentiation among production systems and sites. Compared to integrated apples, organic apples had significantly higher frequencies of filamentous fungi, abundance of total fungi, and taxon diversity. The effects of the production system on the fungal microflora are most likely due to the different plant protection strategies. The incidence of potential mycotoxin producers such as Penicillium and Alternaria species was not different between production systems. We suggest that higher fungal diversity may generally be associated with organic production and may increase the level of beneficial and antagonistically acting species known for their potential to suppress apple pathogens, which may be an advantage to organic apples, e.g., in respect to natural disease control.     

Uptake,demand and internal cycling of nitrogen in saplings of Mediterranean<Emphasis Type="Italic"> Quercus</Emphasis> species

Nitrogen uptake, nitrogen demand and internal nitrogen cycling were studied to address the question of the importance of nutrient storage in Quercus species with contrasting leaf longevities. We carried out this study at the whole-plant level with young trees (2-4 years old) of three Mediterranean Quercus species: the evergreen Q. ilex, the marcescent/evergreen Q. faginea, and the deciduous Q. pyrenaica. Seasonal dynamics of nitrogen in all compartments of the plant were followed for 3 years. Nitrogen losses were measured through litter production, herbivory and fine root shedding. Nitrogen uptake was estimated using increments of nitrogen plant content plus accumulative nitrogen losses. Nitrogen uptake was limited to a few months during late winter and spring. Before budbreak, acquired nitrogen was stored in old-leaf cohorts of evergreen and woody compartments. After budbreak, Quercus species relied first on soil uptake and second on nitrogen retranslocation to supply new growth requirements. However, in most cases we found a high asynchrony between nitrogen demand by growing tissues and soil supply, which determined a strong nitrogen retranslocation up to 88.4% of the nitrogen demand throughout leaf expansion. Except for the first year after planting, the above- and underground woody fractions provided more nitrogen to the new tissues than the old leaf cohorts. Differences in the benefit of nitrogen withdrawn from senescent and old leaves were not found between species. We conclude that sink/source interaction strength was determined by differences between nitrogen demand and uptake, regulating internal nutrient cycling at the whole plant level.     

Allometry of within-fruit reproductive allocation in subtropical dicot woody species

Angiosperm fruits typically consist of pericarp and seed, which collectively function to maximize plant reproductive success. Within-fruit reproductive allocation has been scarcely examined across a wide range of fruit types and taxa although it is critical to the understanding of the evolution of fruit size and seed size. We investigated seed size, fruit size, seed number per fruit (SNF), and within-fruit biomass allocation between seed mass and pericarp mass for 62 dicot woody species (27 deciduous and 35 evergreen species) of a subtropical evergreen forest in southwest China. At the fruit level, total pericarp mass (TPM) isometrically scaled with increasing total seed mass (TSM) in the evergreen species and in the pooled data set, while TPM increased faster than TSM in the deciduous species. The slope difference is possibly due to the difference in the timing of fruit development between the two species groups. At the seed level, seed package (pericarp mass per seed) isometrically scaled with increasing seed size in the deciduous group, but less than isometrically in the evergreens and in the pooled data set. SNF was negatively correlated with seed size but positively correlated with the proportion of pericarp within fruits. In conclusion, within-fruit biomass allocation is significantly affected by seed size, fruit size, and SNF in both deciduous and evergreen species. The implications of the observed scaling relationships are discussed in relation to seed size evolution and global patterns of seed size variation.     

Fungal populations of the haulm base of wheat

Abstract In a 2-year field survey, the phylloplane mycoflora on the haulm base of wheat was studied by the dilution plate technique. No significant colonisation was registered before the end of May. Cladosporium spp., 'white' and 'red' yeasts were found to be the most abundant fungi.     

Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland

Plant host species is considered an important factor influencing ectomycorrhizal (EM) communities. To gain insights into the role of host species in structuring EM communities, EM communities on sympatric oak (Quercus) species were compared in the Sierra Nevada foothills of California. Using molecular methods (polymerase chain reaction, cloning, restriction fragment length polymorphism and DNA sequencing), EM fungi on roots of deciduous Quercus douglasii and evergreen Quercus wislizeni trees were identified from 64 soil cores. The total EM species richness was 140, of which 40 taxa were detected on both oak hosts. Greater diversity and frequency of EM fungi with epigeous fruiting habit were found on Q. wislizeni, while taxa in the Ascomycota were more frequent and diverse on Q. douglasii. Using ordination, it was determined that both soil extractable phosphorus and oak host species explained a significant proportion of the variation in EM species distribution. These results indicate that plant host species can be an important factor influencing EM fungal community composition, even within congeneric trees.     

Diversity and antimicrobial activities of the fungal endophyte community associated with the traditional Brazilian medicinal plant Solanum cernuum Vell. (Solanaceae)

The diversity and antimicrobial activity of endophytic fungi associated with the Brazilian medicinal plant Solanum cernuum Vell. were studied during summer and winter seasons. A total of 246 fungal isolates were obtained, including 225 filamentous fungi and 21 yeasts. They were identified by morphological, physiological, and molecular methods. Fifty-five different taxa represented by the phyla Ascomycota (33 taxa), Basidiomycota (21 taxa), and Zygomycota (one taxon) were identified. The most abundant taxa were closely related to Arthrobotrys foliicola , Colletotrichum gloeosporioides , Coprinellus radians , Glomerella acutata , Diatrypella frostii , Phoma glomerata , Mucor sp., Phlebia subserialis , Phoma moricola , Phanerochaete sordida , and Colletotrichum sp. A total of 265 fungal extracts were screened and 64 (26.01%) displayed antimicrobial activities. Among these extracts, 18 (28.12%) presented antibacterial and antifungal activities, 42 (65.62%) displayed selective antibacterial activity, and four (6.25%) exhibited only antifungal activity. The best values of minimum inhibitory concentration were obtained from extracts of Cryptococcus rajasthanensis , Glomerella acutata, Leptosphaeria sp., and Phoma glomerata ranging from 7.8 to 15.62?μg/mL. This study is the first survey of the endophytic fungi community associated with S. cernuum, and our results show that they can represent a promising source of bioactive compounds.     

Topography and soil content contribute to plant community composition and structure in subtropical evergreen-deciduous broadleaved mixed forests

Topography and soil factors are known to play crucial roles in the species composition of plant communities in subtropical evergreen-deciduous broadleaved mixed forests. In this study, we used a systematic quantitative approach to classify plant community types in the subtropical forests of Hubei Province (central China), and then quantified the relative contribution of drivers responsible for variation in species composition and diversity. We classified the subtropical forests in the study area into 12 community types. Of these, species diversity indices of three communities were significantly higher than those of others. In each community type, species richness, abundance, basal area and importance values of evergreen and deciduous species were different. In most community types, deciduous species richness was higher than that of evergreen species. Linear regression analysis showed that the dominant factors that affect species composition in each community type are elevation, slope, aspect, soil nitrogen content, and soil phosphorus content. Furthermore, structural equation modeling analysis showed that the majority of variance in species composition of plant communities can be explained by elevation, aspect, soil water content, litterfall, total nitrogen, and total phosphorus. Thus, the major factors that affect evergreen and deciduous species distribution across the 12 community types in subtropical evergreen-deciduous broadleaved mixed forests include elevation, slope and aspect, soil total nitrogen content, soil total phosphorus content, soil available nitrogen content and soil available phosphorus content.     

Positional variation in phylloplane microbial populations within an apple tree canopy

Variation in density of epiphytic yeasts, filamentous fungi, and bacteria on apple leaves collected from eight trees at nine dates for two seasons was determined with respect to three positional factors: height, compass direction from the center of the tree, and lateral proximity to the canopy periphery. Univariate analyses of variance were performed on each of the microbial classes for each date according to a model that excluded tree effect but accounted for the positional factors with interactions. The assumption of no tree effect was explored by residual analysis and examination of the seasonal pattern of microbial densities for each tree. No persuasive evidence was obtained to invalidate this assumption. For filamentous fungi and yeasts, height and lateral position were the most significant factors withp<0.05 for yeasts at several periods. The two factors appeared to be of equal importance. Trends were less clear for bacteria, but all three positional factors and some two-way interactions seemed of some importance. For filamentous fungi and bacteria, frequently no factors were significant at a level of 0.10, but at almost all sampling dates certain positional factors and interactions were significant at a level of 0.25. Inspection of partial correlation coefficients indicated no apparent linear association between densities of most pairs of microbial classes. Implications of these results for experimental design and for the microbial ecology of the phylloplane community are discussed.     

On the ecology of fungal consortia of spring sap-flows

Spring sap-flow, i.e. xylem sap exuding through wounds of deciduous trees in spring, quickly becomes colonised by yeasts and filamentous fungi, of which several species occur regularly in consortia from different trees or regions. This article considers the survival, spread and population dynamics of these fungi, with particular emphasis on the substantial research by Wladyslav I. Golubev on birch sap-flows in Russia. Most fungi survive in the soil or epiphytically on tree trunks, and dispersal between trees is probably mediated by insects. Competition within and between sap-flow yeast species may be due to nutritional effects or the action of killer toxins (mycocins).     

Phylloplane fungi in Hong Kong mangroves: evaluation of study methods

Many methods have been used to study phylloplane fungi, most of which have constraints and may result in biased results. This study used light microscopy and scanning electron microscopy (SEM) to investigate fungal abundance on the leaves of the most common mangrove trees in Hong Kong, Kandelia candel and Aegiceras corniculatum. Species richness was investigated using light microscopy and a leaf washing method. Methods to study phylloplane fungi are discussed and the performances of these three investigation methods are evaluated. Seven mitosporic fungal taxa were found by light microscopy, while 30 sporulating taxa and 18 Mycelia sterilia were isolated using the leaf washing method. Fungal abundance in terms of percentage cover investigated with light microscopy was similar using the SEM method, and was significantly higher on Aegiceras corniculatum than on Kandelia candel. Fungal abundance peaked in the summer and was lowest in the winter. This study indicates that light microscopy reveals the most typical phylloplane fungi and is more efficient than SEM, while the leaf washing method reveals many casual species and is not quantitative.     

Differential and interactive effects of temperature and photoperiod on budburst and carbon reserves in two co-occurring Mediterranean oaks

Effects of temperature and photoperiod and their interactions on budburst and on the use of carbon reserves were examined in two Mediterranean oaks differing in wood anatomy and leaf habit. Seedlings of Quercus ilex subsp. ballota (evergreen and diffuse-porous wood) and Q. faginea (semi-deciduous and ring-porous wood) were grown under two temperatures (12 and 19 °C) and two photoperiods (10 and 16 h) in a factorial experiment. In the 16 h photoperiod at 19 °C, photosynthesis was suppressed in half of the seedlings by covering leaves with aluminium foil. The concentration of soluble sugars, starch and lipids in leaves, stems and roots was assessed before and after budburst. Under the 12 °C treatment (mean current temperature in early spring in the Iberian Peninsula), budburst in Q. faginea occurred earlier than in Q. ilex . Higher temperature promoted earlier budburst in both species, mostly under the 16 h photoperiod. This response was less pronounced in Q. faginea because its budburst was also controlled by photoperiod, and because this species needs to construct a new ring of xylem before budburst to supply its growth demands. Therefore, dates of budburst of the two species became closer to each other in the warmer treatment, which might alter competitive relations between the species with changing climate. While Q. ilex relied on carbon reserves for budburst, Q. faginea relied on both carbon reserves and current photoassimilates. The different responses of the two Quercus species to temperature and photoperiod related more to xylem structure than to the source of carbon used for budburst.     

Leaf Mass per Area (LMA) and Its Relationship with Leaf Structure and Anatomy in 34 Mediterranean Woody Species along a Water Availability Gradient

Leaf mass per area (LMA) is a morphological trait widely used as a good indicator of plant functioning (i.e. photosynthetic and respiratory rates, chemical composition, resistance to herbivory, etc.). The LMA can be broken down into the leaf density (LD) and leaf volume to area ratio (LVA or thickness), which in turn are determined by anatomical tissues and chemical composition. The aim of this study is to understand the anatomical and chemical characteristics related to LMA variation in species growing in the field along a water availability gradient. We determined LMA and its components (LD, LVA and anatomical tissues) for 34 Mediterranean (20 evergreen and 14 deciduous) woody species. Variation in LMA was due to variation in both LD and LVA. For both deciduous and evergreen species LVA variation was strongly and positively related with mesophyll volume per area (VA or thickness), but for evergreen species positive relationships of LVA with the VA of epidermis, vascular plus sclerenchyma tissues and air spaces were found as well. The leaf carbon concentration was positively related with mesophyll VA in deciduous species, and with VA of vascular plus sclerenchymatic tissues in evergreens. Species occurring at the sites with lower water availability were generally characterised by a high LMA and LD.     

Quantitative and qualitative studies of the microflora of barley malt production

Populations of aerobic heterotrophic bacteria, mycelial fungi and yeasts occurring in malting barley were estimated by a plate technique and scanning electron microscopy. There was an increase in the total number of micro-organisms during germination, although populations declined after kilning. Bacteria dominated on all samples, with progressively lower populations of yeasts and filamentous fungi. There was no obvious spatial distribution of micro-organisms on the samples although there appeared to be high populations of bacteria and fungal hyphae on the inner surface of the kernels. The dominant groups of aerobic heterotrophic bacteria were presumptively identified as Alcaligenes sp., Arthrobacter globiformis, Clavibacter iranicum, Erwinia herbicola, Lactobacillus spp. and Pseudomonas fluorescens. The principal filamentous fungi were identified as Aiternaria alternata, Aspergillus glaucus (group), Cladosporium macrocarpum, Epicoccum purpurascens, Fusarium avenaceum, Geotrichum candidum and Penicillium spp. The yeasts isolated most frequently were Candida catenulata, C. vini, Debaryomyces hansenii, Hansenula polymorpha, Kloeckera apiculata, Rhodotorula mucilaginosa, Sporobolomyces roseus and Trichosporon beigelii.     

Tests of the antibiotic properties of the invasive vine Vincetoxicum rossicum against bacteria, fungi and insects

We tested extracts of Vincetoxicum rossicum for inhibition of bacterial and fungal growth and for anti-insect activities that may account for the invasive characteristics of this introduced species in North America. Bioassay-guided fractionation was used to identify (−)-antofine as the principle inhibitor of bacteria and fungi in root extracts. This compound had especially pronounced antifungal activity, inhibiting the growth of diverse taxa that include yeast-like and filamentous fungi and, notably, broad-host-range plant pathogens. A second compound(s), that is as yet uncharacterized but distinct from (−)-antofine, was detected as having antifeedant activity against a larval hymenopteran, rose sawfly (Allantus cinctus), and toxicity to two larval lepidopterans, the masked birch caterpillar (Drepana arcuata) and the European corn borer (Ostrinia nubilalis). That V. rossicum contains potent inhibitors of plant pathogenic fungi, diverse bacteria, and herbivorous insects likely contributes to its success as an invasive species.     

Experimental warming alters potential function of the fungal community in boreal forest

Fungal community composition often shifts in response to warmer temperatures, which might influence decomposition of recalcitrant carbon (C). We hypothesized that evolutionary trade‐offs would enable recalcitrant C‐using taxa to respond more positively to warming than would labile C‐using taxa. Accordingly, we performed a warming experiment in an Alaskan boreal forest and examined changes in the prevalence of fungal taxa. In a complementary field trial, we characterized the ability of fungal taxa to use labile C (glucose), intermediate C (hemicellulose or cellulose), or recalcitrant C (lignin). We also assigned taxa to functional groups (e.g., free‐living filamentous fungi, ectomycorrhizal fungi, and yeasts) based on taxonomic identity. We found that response to warming varied most among taxa at the order level, compared to other taxonomic ranks. Among orders, ability to use lignin was significantly related to increases in prevalence in response to warming. However, the relationship was weak, given that lignin use explained only 9% of the variability in warming responses. Functional groups also differed in warming responses. Specifically, free‐living filamentous fungi and ectomycorrhizal fungi responded positively to warming, on average, but yeasts responded negatively. Overall, warming‐induced shifts in fungal communities might be accompanied by an increased ability to break down recalcitrant C. This change in potential function may reduce soil C storage under global warming.     

Isolation and characterization of some mycelia inhabiting Tuber ascomata

Tuber spp. are ectomycorrhizal ascomycetes that produce subterranean ascomata known as truffles. Truffles can be regarded as complex microhabitats hosting bacteria and yeasts. In this paper we show that guest filamentous fungi are also associated to truffle ascomata, regardless of the Tuber spp., and report the morpho-molecular characterization of seven truffle-hosted mycelia isolated from healthy and intact Tuber ascomata. Some of these isolates were shown to be related to the fungal endophytes of plants. Interestingly, the truffle-hosted mycelia grew stuck to the hyphal wall of their partner when co-cultivated with the Tuber borchii mycelium, but not when co-cultivated with the test species Agaricus macrosporus. The present data suggest that guest filamentous fungi can be added to the list of truffle-interacting microorganisms.