Comparison of the Phenotypes and Genotypes of Biofilm and Solitary Epiphytic Bacterial Populations on Broad-Leaved Endive

The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.     

Colonization and lignin decomposition of Camellia japonica leaf litter by endophytic fungi

Endophytic fungi occur on various types of leaf litter, but few studies have been done on their roles as saprophytes in decomposition. This study examined the succession of fungi in live, newly shed, and decomposing leaves at 2 months of decomposition of Camellia japonica and chemical changes in decomposing leaves colonized by endophytes. Coccomyces nipponicum, Lophodermium sp., Geniculosporium sp. 1, and Colletotrichum gloeosporioides were isolated from living leaves. Coccomyces nipponicum and Lophodermium sp. were also isolated frequently from newly shed and decomposing leaves. These two fungi caused a decrease of lignin content and bleaching in decomposing leaves under field and laboratory conditions. Total hyphal length in decomposing leaves was higher in bleached portions than in surrounding nonbleached portions, which probably reflected the early onset of hyphal growth of endophytes inside leaf tissue at leaf senescence or death. Incubation of newly shed leaves that were sterilized to exclude previously established endophytes resulted in no occurrence of bleached portions in decomposing leaves on the forest floor. This result indicated that these endophytes were incapable of colonizing leaves directly after litterfall and that the persistence of endophytes from live leaves was crucial for their colonization in decomposing leaves.     

Antimicrobial activity of essential oils of Thymus vulgaris and Origanum vulgare on phytopathogenic strains isolated from soybean

The aim of this work was to study the antimicrobial activity of essential oils obtained from Thymus vulgaris (thyme) and Origanum vulgare (oregano) on phytopathogenic Pseudomonas species isolated from soybean. Strains with characteristics of P. syringae were isolated from leaves of soybean plants with blight symptoms. Ten of these could be identified in Group Ia of LOPAT as P. syringae. Six of these were confirmed as P. syringae using 16S rRNA, indicating the presence of these phytopathogenic bacteria in east and central Argentina. All the phytopathogenic bacteria were re‐isolated and identified from the infected plants. MIC values for thyme were 11.5 and 5.7 mg·ml^?1 on P. syringae strains, while oregano showed variability in the inhibitory activity. Both essential oils inhibited all P. syringae strains, with better inhibitory activity than the antibiotic streptomycin. The oils were not bactericidal for all pseudomonads. Both oils contained high carvacrol (29.5% and 19.7%, respectively) and low thymol (1.5%). Natural products obtained from aromatic plants represent potential sources of molecules with biological activity that could be used as new alternatives for the treatment of phytopathogenic bacteria infections.     

The effects of herbicides and mycoparasites at different moisture levels on carpogenic germination in Sclerotinia sclerotiorum

Colonization of plant roots by fluorescent pseudomonads has been correlated with disease suppression. One mechanism may involve altered defense responses in the plant upon colonization. Altered defense responses were observed in bean (Phaseolus vulgaris) inoculated with fluorescent pseudomonads. Systemic effects of root inoculation by Pseudomonas putida isolate Corvallis, P. tolaasii (P9A) and P. aureofaciens REW1-I-1 were observed in bean leaves from 14-day-old plants. SDS- polyacrylamide gel electrophoresis demonstrated that levels of certain acid-soluble proteins increased in the leaf extracts of inoculated plants. Plants inoculated with REW1-I-1 produced more of a 57 M_r protein, and plants inoculated with isolates P9A and REW1-I-1 produced more of a 38 M_r protein. Northern hybridization revealed enhanced accumulation of mRNAs, that encode the pathogenesis-related protein PR1a, in leaves of plants inoculated with P. putida and REW1-I-1. Only REW1-I-1, but not P9A or P. putida induced symptoms of an hypersensitive response on tobacco leaves, bean cotyledons, and in bean suspension cultures. Phenolics and phytoalexins accumulated in bean cotyledons exposed to REW1-I-1 for 24 h but little change in levels of these compounds occurred in cotyledons inoculated with P9A and P. putida. Both suspension culture cells and roots treated with REW1-I-1 rapidly evolved more hydrogen peroxide than those exposed to P9A and P. putida. However, roots from 14-day-old plants colonized by P9A, P. putida or REW1-I-1 did not have higher levels of phenolics, phytoalexins or mRNAs for two enzymes involved in phenolic biosynthesis, phenylalanine-ammonia lyase and chalcone synthase. A selective induction of plant defense strategies upon root colonization by certain pseudomonads is apparent.     

The Fungal and Bacterial Flora of Stored White Cabbage

1. The predominant organisms isolated from the outer wrapper leaves of freshly harvested white cabbages were: bacteria, yeasts, Alternaria spp., Aureobasidium pullulans, Botrytis cinerea, Cladosporium spp. and Penicillium spp.
2. Few qualitative or quantitative changes were seen in the leaf surface flora during storage at 2°C for up to 33 weeks.
3. Numbers of bacteria, particularly fluorescent and pectolytic pseudomonads, were considerably higher on cabbages drenched with fungicide or water than on corresponding undrenched cabbages.     

Grapevine leaf gas exchange, plant growth, yield, fruit quality and carbohydrate reserves influenced by the grape leafhopper,Empoasca vitis

The impact of the grape leafhopper,Empoasca vitis, on leaf gas exchange, plant growth, yield, fruit quality and carbohydrate reserves of the grapevines,Vitis vinifera L., was studied. Gas exchange was measured on the discolored (red) and the green parts of infested main leaves and on leaves from uninfested vines. Photosynthesis and mesophyll conductance were severely reduced on main leaves showing leafhopper feeding symptoms. The stomatal conductance of the red leaf section of infested main leaves was lower than on undamaged control leaves. Additionally, the red leaf section of infested main leaves showed lower transpiration rates when compared to the green parts of the same leaves and to undamaged control leaves. Gas exchange processes of lateral leaves were not affected by leafhopper feeding. Leafhopperload on main leaves was correlated to visual damage symptoms. At 71.8 leafhopper-days per leaf up to 40% of the main leaf area of the infested plants was discolored from the borders towards the center. Lateral leaves showed no feeding symptoms. Shoot diameter, pruning weight and carbohydrate reserves in the wood were not affected by leafhoppers. Lateral leaf area growth was significantly stimulated on plants infested by leafhoppers. No decrease in yield and fruit quality with leafhopper-loads up to 71.8 leafhopper-days per leaf were observed.     

Inoculum Sources for the Spread of Leaf Scald Disease of Sugarcane Caused by Xanthomonas albilineans in Guadeloupe

Leaf scald of sugarcane, caused by Xanthomonas albilineans, is thought to be spread mainly in infected cuttings and transmitted on infested cutting implements. Several observations made in Guadeloupe indicated that other means of spreading also occur. The dispersal of the pathogen outside sugarcane was investigated with plants inoculated by an antibiotic-resistant marked strain of X. albilineans and with plants naturally infested with wild strains of the pathogen. The bacteria were isolated in water droplets (rain or dew) on the surface of sugarcane leaves at dawn. It was also detected on the surface of dry leaves during the day by leaf imprinting onto a selective culture medium. The bacteria were much more frequently isolated from the surface of symptomatic leaves than from symptomless ones. Aerial dispersal of X. albilineans was investigated by placing Petri dishes containing selective culture medium between sugarcane plants but without direct contact with the leaves. The pathogen was isolated in four out of 270 dishes which were randomly set 3–14 h in a diseased field. These results indicated that the pathogen exuded from the leaves and then was spread by aerial means (rain, insects, …) or by leaf contact. The bacteria were also found in roots and rhizospheric soil of infested sugarcane stools suggesting that X. albilineans could be transmitted by root to root contact or by the soil. Finally, isolations of the pathogen in sugarcane inflorescences were positive. So, fuzz transmission may also occur.     

Inoculum Sources for the Spread of Leaf Scald Disease of Sugarcane Caused by Xanthomonas albilineans in Guadeloupe

Leaf scald of sugarcane, caused by Xanthomonas albilineans, is thought to be spread mainly in infected cuttings and transmitted on infested cutting implements. Several observations made in Guadeloupe indicated that other means of spreading also occur. The dispersal of the pathogen outside sugarcane was investigated with plants inoculated by an antibiotic-resistant marked strain of X. albilineans and with plants naturally infested with wild strains of the pathogen. The bacteria were isolated in water droplets (rain or dew) on the surface of sugarcane leaves at dawn. It was also detected on the surface of dry leaves during the day by leaf imprinting onto a selective culture medium. The bacteria were much more frequently isolated from the surface of symptomatic leaves than from symptomless ones. Aerial dispersal of X. albilineans was investigated by placing Petri dishes containing selective culture medium between sugarcane plants but without direct contact with the leaves. The pathogen was isolated in four out of 270 dishes which were randomly set 3-14 h in a diseased field. These results indicated that the pathogen exuded from the leaves and then was spread by aerial means (rain, insects,…) or by leaf contact. The bacteria were also found in roots and rhizospheric soil of infested sugarcane stools suggesting that X. albilineans could be transmitted by root to root contact or by the soil. Finally, isolations of the pathogen in sugarcane inflorescences were positive. So, fuzz transmission may also occur.     

Pseudomonads associated with midrib rot and soft rot of butterhead lettuce and endive

During the past ten years, bacterial soft rot and midrib rot of glasshouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) and field-grown endive (Cichorium endivia L.) has become increasingly common in the region of Flanders, Belgium. Severe losses and reduced market quality caused by bacterial rot represent an important economical threat for the production sector. Symptoms of midrib rot are a brownish rot along the midrib of one or more inner leaves, often accompanied by soft rot of the leaf blade. Twenty-five symptomatic lettuce and endive samples were collected from commercial growers at different locations in Flanders. Isolations of dominant bacterial colony types on dilution plates from macerated diseased tissue extracts yielded 282 isolates. All isolates were characterized by colony morphology and fluorescence on pseudomonas agar F medium, oxidase reaction, and soft rot ability on detached chicory leaves. Whole-cell fatty acid methyl esters profile analyses identified the majority of isolates (85%) as belonging to the Gammaproteobacteria, which included members of the family Enterobacteriaceae (14%) and of the genera Pseudomonas (73%), Stenotrophomonas (9%), and Acinetobacter (3%). Predominant bacteria were a diverse group of fluorescent Pseudomonas species. They were further differentiated based on the non-host hypersensitive reaction on tobacco and the ability to rot potato slices into 4 phenotypic groups: HR-/P- (57 isolates), HR-/P+ (54 isolates), HR+/P (16 isolates) and HR+/P+ (35 isolates). Artificial inoculation of suspensions of HR-, pectolytic fluorescent pseudomonads in the leaf midrib of lettuce plants produced various symptoms of soft rot, but they did not readily cause symptoms upon spray inoculation. Fluorescent pseudomonads with phenotype HR+ were consistently isolated from typical dark midrib rot symptoms, and selected isolates reproduced the typical midrib rot symptoms when spray-inoculated onto healthy lettuce plants.     

Characterization of Pseudomonas fuscovaginae and Differentiation from Other Fluorescent Pseudomonads Occurring on Rice in Burundi

In various pathogenicity, serological, physiological and biochemical tests, performed in Belgium and Japan, the Pseudomonas fuscovaginae strains associated above 1,350 m elevation in Burundi with sheath brown rot of rice, rusty seed and black, stripes on seedlings, were found to be similar to reference strains of this pathogen from Japan. The same bacteria was detected on rice seeds imported from Asia to Burundi. Beside the serological characteristics, P. fuscovaginae can be differentiated from other oxidase and arginine dihydrolase positive non-pathogenic fluorescent pseudomonads, also isolated from lesions on rice seedlings, by the simultaneous occurrence of no production of 2-ketogluconate, acid production from trehalose, but not from inositol. Occasionally, other symptoms inducing, oxidase positive, fluorescent pseudomonads, different from any described species, were isolated from rice seedlings and sheath rot in Burundi.     

Biological control of winter wheat pathogens with the use of antagonistic Sphingomonas bacteria under greenhouse conditions

This paper describes, for the first time, the effect of bacteria of the genus Sphingomonas on healthiness of winter wheat. The effect of the application of Sphingomonas cell suspension on development of disease symptoms of powdery mildew and Fusarium head blight (FHB) of winter wheat cv. Bogatka was studied under greenhouse conditions. The abundance of populations of yeast and fungi producing mycelium as well as bacteria of the genus Azotobacter and pseudomonads was determined on wheat kernels. The biocontrol agent reduced the population size of Fusarium poae, and it contributed to better grain filling. The tested Sphingomonas isolate reduced the severity of flag leaf infection caused by pathogenic biotroph Blumeria graminis f. sp. tritici.     

Interactions Between Hyphosphere-Associated Bacteria and the Fungus <Emphasis Type="Italic">Cladosporium herbarum</Emphasis> on Aquatic Leaf Litter

We investigated microbial interactions of aquatic bacteria associated with hyphae (the hyphosphere) of freshwater fungi on leaf litter. Bacteria were isolated directly from the hyphae of fungi from sedimented leaves of a small stream in the National Park “Lower Oder,” Germany. To investigate interactions, bacteria and fungi were pairwise co-cultivated on leaf-extract medium and in microcosms loaded with leaves. The performance of fungi and bacteria was monitored by measuring growth, enzyme production, and respiration of mono- and co-cultures. Growth inhibition of the fungus Cladosporium herbarum by Ralstonia pickettii was detected on leaf extract agar plates. In microcosms, the presence of Chryseobacterium sp. lowered the exocellulase, endocellulase, and cellobiase activity of the fungus. Additionally, the conversion of leaf material into microbial biomass was retarded in co-cultures. The respiration of the fungus was uninfluenced by the presence of the bacterium.     

Isolation and characterization of heavy metals resistant bacteria from Lagos Lagoon

A total of 228 bacteria with an ability to resist toxic heavy metals were isolated from 8 selected sites of the Lagos Lagoon. The bacteria isolated wereStaphylocaccus sp.,Bacillus sp.,Pseudomonas sp.,Streptococcus sp.,Moraxella sp.,Escherichia coli, Proteus sp.,Klebsiella sp. andSalmonella sp. The heavy metals to which resistance was recorded were mercury, lead, zinc, cobalt, copper and chromium. The lagoon sites from which the highest number of resistant bacteria were isolated were Marina and Ebute-Ero. The heavy metal to which most bacteria were resistant was cobalt, while the least was chromium. The significance of the result is discussed in relation to the Nigerian environment and human health.     

The bacterial microflora of witloof chicory (Cichorium intybus L. var.foliosum Hegi) leaves

The bacterial flora on the heads of four different witloof chicory varieties was examined. The 590 isolates were characterized by their SDS-PAGE protein profiles; they revealed 149 different protein fingerprint types. The fluorescentPseudomonas fingerprint type CH001 was abundantly found on all heads examined. Fourteen other fingerprint types occurred in high densities more than twice. Among these, the following were identified: fluorescentPseudomonas, nonfluorescentPseudomonas sp.,Erwinia herbicola, Erwinia sp., andFlavobacterium sp. The majority of the fingerprint types (90%) was found only once. It was also our objective to isolate bacteria applicable in the biological control of chicory phytopathogens. Isolates of all fingerprint types were tested for in vitro antagonistic activity and for possible deleterious effect on plant growth. FluorescentPseudomonas andSerratia liquefaciens isolates were antagonistic against fungi. Among the 161 fluorescentPseudomonas strains, five were able to produce disease symptoms on chicory leaves upon inoculation. Comparison of the results of this study with those obtained in two previous analyses revealed that the leaf microflora showed some similarities with the bacterial flora of chicory roots. The chicory seed microflora differed from that of both leaves and roots.     

The effects of a pesticide program on microbial populations from apple leaf litter

The leaf litter microbial community was quantitatively and qualitatively changed when a standard pesticide schedule that comprised an insecticide, a bactericide, and a fungicide was applied to McIntosh apple trees in the summer. Effects were observed for two winters by four indirect assays and three direct methods. Populations were altered qualitatively both years, but the most striking difference was the quantitative impact from year to year. Bacteria, filamentous fungi, and yeasts from treated leaves were reduced 10- to 10 000-fold between November 1976 and April 1977 and did not recover until snow cover had melted in March. Reductions in 1977-1978 were negligible. The marked seasonal difference is attributed to meteorological influences. Fluorescent pseudomonads were among the bacteria depressed by chemicals. Of the 49 genera of fungi and yeasts isolated, Coniothyrium sp., Penicillium spp., Arthrobotrys spp., and Nodulisporium sp. were appreciably reduced, whereas Typhula spp., Pleurophomella sp., Sporobolomyces spp., and Rhodotorula spp. were substantially enhanced by the spray program.     

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.     

The ppuI-rsaL-ppuR quorum-sensing system regulates cellular motility,pectate lyase activity,and virulence in potato opportunistic pathogen Pseudomonas sp. StFLB209

Pseudomonas sp. StFLB209 was isolated from potato leaf as an N-acylhomoserine lactone (AHL)-producing bacterium and showed a close phylogenetic relationship with P. cichorii, a known plant pathogen. Although there are no reports of potato disease caused by pseudomonads in Japan, StFLB209 was pathogenic to potato leaf. In this study, we reveal the complete genome sequence of StFLB209, and show that the strain possesses a ppuI-rsaL-ppuR quorum-sensing system, the sequence of which shares a high similarity with that of Pseudomonas putida. Disruption of ppuI results in a loss of AHL production as well as remarkable reduction in motility. StFLB209 possesses strong pectate lyase activity and causes maceration on potato tuber and leaf, which was slightly reduced in the ppuI mutant. These results suggest that the quorum-sensing system is well conserved between StFLB209 and P. putida and that the system is essential for motility, full pectate lyase activity, and virulence in StFLB209.     

Probable mechanical transmission of a virus-like agent from rose rosette disease-infected multiflora rose to Nicotiana species

As part of efforts to identify the causal agent of the rose rosette disease (RRD) of multiflora rose (Rosa multiflora Thunb.), root tip extracts from both symptomatic and nonsymptomatic roses were used to mechanically inoculate leaves of Nicotiana glutinosa. Pale green spots were observed along the margins of the major leaf veins only on leaves inoculated with extracts prepared from symptomatic rose plants. Light microscopy revealed abnormal development of the palisade and spongy mesophyll cells in the symptomatic tissue, although no virus‐like particles (VLPs) were observed by electron microscopy. However, VLPs were observed in cells from tissue adjacent to the leaf veins and bordered by the pale green spots. Inoculation of N. benthamiana with extracts from symptomatic N. glutinosa initially did not result in visible symptoms on N. benthamiana inoculated leaves. However, approximately 4 wk post inoculation, splitting of leaf tissue across and along major leaf veins in expanding leaves occurred. In later stages of leaf expansion some leaves split in regions not associated with veins. Light microscopy of thick sections revealed separation between palisade cells and groups of small dead cells in the mesophyll tissue of expanding systemically infected leaf blades. Electron microscopy revealed crystalline arrays in the cytoplasm of mesophyll cells. No abnormal cellular changes were observed in plants inoculated with extracts prepared from nonsymptomatic rose plants.     

Consequences of the colonisation of leaves by fungi and oomycetes for leaf consumption by a gammarid shredder

1. Leaf litter breakdown by shredders in the field is affected by leaf toughness, nutritional value and the presence of secondary compounds such as polyphenols. However, experiments involving the use of single fungal strains have not supported the assumption that leaf parameters determine food selection by shredders perhaps because of a failure to test for high consumption prior to isolation of fungal strains, overrepresentation of hyphomycetes or the potential effects of accompanying bacteria. In this study, we used bacteria‐free, actively growing fungi and oomycetes isolated from conditioned leaf litter for which a shredder had already shown high consumption rates. 2. Black alder (Alnus glutinosa) leaf litter was exposed to the littoral zone of Lake Constance in autumn, and subsamples were analysed for leaf parameters and consumption by Gammarus roeselii under standard conditions at regular intervals. On dates with a high consumption rate of the exposed leaves, 14 single strains of fungi and oomycetes were isolated, freed of bacteria and grown on autoclaved leaves. 3. Six of eight measured leaf parameters of exposed leaves were significantly correlated with Gammarus consumption rates, with high colinearity among leaf parameters hampering the identification of causal relations between leaf parameters and feeding activity. 4. When single strains of fungi and oomycetes were grown on autoclaved leaf litter, toughness of colonised leaves was always lower than in the control and the content of protein, N and P were increased. There were pronounced strain‐specific effects on leaf parameters. Consumption rates also differed significantly, with nine of fourteen isolates consumed at higher rates than controls and none proving to be a deterrent. Protein and polyphenol content were significantly correlated with consumption rates. Oomycete‐colonised leaves were consumed at similar rates but were of lower food quality than fungi‐colonised leaves. 5. We argue that direct strain‐specific attractant or repellent effects of fungi and oomycetes on consumption by G. roeselii are not important. However, we found indirect strain‐specific role operating via effects on leaf parameters.     

Seasonal and leaf age-dependent changes in occurrence of phyllosphere fungi of giant dogwood

To examine the relative importance of leaf age and season on the occurrence of phyllosphere fungi, temporal patterns of epiphytic and endophytic phyllosphere fungi of giant dogwood (Swida controversa) were studied with reference to leaf emergence at first occurrence and in the middle of the growing season. A total of 15 and 44 species were isolated from the surface and interior of leaves, respectively. On the leaf surface, detection rate of fungi was consistently 100% and their frequencies increased during the growing season, whereas in the leaf interior, detection rate of fungi and their frequencies were low at leaf emergence and gradually increased during the growing season. Six epiphytic and two endophytic fungi were observed frequently. A white sterile mycelium was frequent only on the surface of newly emerged leaves in the first-order shoot in May. The other 7 species increased during the growing season. The frequencies of Phomopsis sp., Pestalotiopsis sp. 1, and Trichoderma viride were higher on the leaves of first-order shoots than those of higher-order shoots that emerged between July and September, suggesting the possible effects of leaf age on their occurrence. On the other hand, the frequencies of Colletotrichum gloeosporioides, Clonostachys rosea, Cladosporium cladosporioides, and Phoma sp. 1 were not different between the first- and higher-order shoots, suggesting the negligible effect of leaf age. The influence of phenological patterns of leaf emergence of deciduous trees on the diversity and composition of assemblages of phyllosphere fungi is discussed.