Stauro-and scolecoconidia in floral and honeydew honeys

Abstract

Forty-four samples of floral and honeydew honeys from Croatia, Greece, Italy, Mexico, New Zealand, Portugal, South Africa, Spain and Tanzania were microscopically examined for fungal spores. Most of the floral honeys were dominated by yeast cells of Metschnikowia reukaufii and contained very few conidia of hyphomycete species. By contrast, honeydew honeys contained scoleco-and stauroconidia belonging to more than 30 hyphomycete species, most of them previously reported from rainwater on living trees. Most belonged to the genera Tripospermum, Retiarius and Trinacrium. Their concentrations were highest in the honeydew honey from Abies alba and Picea excelsa. Conidia belonging to species of Camposporium, Ceratosporium, Dwayaangam, Tricellula, Tricladium and Trifurcospora, well-known litter-inhabiting fungi in terrestrial and/or aquatic habitats were encountered. Some other conidia probably belonged to species of Articulospora, Curucispora, Gyoerffyella, Lemonniera and Varicosporium, also well-known Ingoldian fungi from lotic ecosystems. The assemblages of fungal spores in honeydew honeys may provide important information on the geographical distribution of "canopy fungi". In addition, the results of this study support Carroll's theory on the existence of a fungal group termed "arboreal aquatic hyphomycetes" or "canopy fungi". Although their function in canopies is presently unknown, evidence accumulating in the literature suggests their widespread occurrence in the phyllosphere.

Full text

Fungal Diversity
Stauro- and scolecoconidia in floral and honeydew honeys
Magyar D.1*, Gönczöl J.2, Révay Á.2, Grillenzoni F.3 and Seijo-Coello
M.D.C.4
1Plant Protection Institute of the Hungarian Academy of Sciences, Department of Plant
Pathology, H-1525 Budapest, Pf. 102, Hungary
2Hungarian Natural History Museum, Botanical Department, H-1476 Budapest, Pf. 222,
Hungary.
3National Institute for Apiculture, Via di Saliceto, 80 40128 Bologna, Italy.
4University of Vigo, Faculty of Science, Department of Vegetal Biology and Soil Science,
Campus as Lagoas, 32004, Ourense, Spain
Magyar, D., Gönczöl, J., Révay, Á., Grillenzoni, F. and Seijo-Coello, M.D.C. (2005). Stauro-
and scolecoconidia in floral and honeydew honeys. Fungal Diversity 20: 103-120.
Forty-four samples of floral and honeydew honeys from Croatia, Greece, Italy, Mexico, New
Zealand, Portugal, South Africa, Spain and Tanzania were microscopically examined for
fungal spores. Most of the floral honeys were dominated by yeast cells of Metschnikowia
reukaufii and contained very few conidia of hyphomycete species. By contrast, honeydew
honeys contained scoleco- and stauroconidia belonging to more than 30 hyphomycete species,
most of them previously reported from rainwater on living trees. Most belonged to the genera
Tripospermum, Retiarius and Trinacrium. Their concentrations were highest in the honeydew
honey from Abies alba and Picea excelsa. Conidia belonging to species of Camposporium,
Ceratosporium, Dwayaangam, Tricellula, Tricladium and Trifurcospora, well-known litter-
inhabiting fungi in terrestrial and/or aquatic habitats were encountered. Some other conidia
probably belonged to species of Articulospora, Curucispora, Gyoerffyella, Lemonniera and
Varicosporium, also well-known Ingoldian fungi from lotic ecosystems. The assemblages of
fungal spores in honeydew honeys may provide important information on the geographical
distribution of "canopy fungi". In addition, the results of this study support Carroll's theory on
the existence of a fungal group termed "arboreal aquatic hyphomycetes" or "canopy fungi".
Although their function in canopies is presently unknown, evidence accumulating in the
literature suggests their widespread occurrence in the phyllosphere.
Key words: "canopy fungi", floral honey, honeydew honey, phyllosphere, stauro- and
scolecoconidia.
Introduction
The existence of aquatic hyphomycetes in terrestrial forest litter has
received little attention and even less so on living plants. There are, however, a
* Corresponding author: D. Magyar; e-mail: magyard@freemail.hu
103

few papers recording conidia of aquatic hyphomycetes in "unexpected places"
(Carroll, 1981). Samples of throughfall from Douglas fir trees (Pseudotsuga
menziesii (Mirb.) Franco) revealed numerous triradiate and tetraradiate conidia
belonging to genera such as Tripospermum, Tridentaria and Ceratosporium
amongst others. It was thought that these constituted a guild ("arboreal aquatic
hyphomycetes" or "canopy fungi") which "may function in canopies much as
classical aquatic hyphomycetes function in streams" (Carroll, 1981). Bandoni
(1981) reported conidia of Gyoerffyella biappendiculata, G. gemellipara and
Tripospermum spp. from stemflow of several trees in British Columbia,
Canada. Ando and Tubaki (1984a, b) recorded conidia of aquatic
hyphomycetes and described new species and genera from throughfall
collected from various trees in Japan. They stated that these fungi "probably
live usually on intact leaves as mycelia and can sporulate when leaves are
moistened by mist, morning dew or rain". Ando (1992) later proposed the term
"terrestrial aquatic hyphomycetes". Gönczöl (1976) reported an assemblage of
mainly stauroconidia from foam also collected in an "unexpected place" i.e. on
the trunk of a beech tree (Fagus sylvatica). In more recent studies Gönczöl and
Révay (2003, 2004) identified conidia of 63 mostly hyphomycete species in
stemflow and throughfall samples from living trees, and 45 hyphomycetes
from treeholes in Hungary.
So far, rainwater is only known as a medium for accumulating and
transporting these fungal spores on trees. During routine melissopalynological
analyses of honeys kept in the National Institute for Apiculture, Bologna, Italy,
fungal spores of unknown identity were encountered, mainly in honeydew
honeys. Some conidia were very similar to those sometimes recorded from
rainwater on live trees. This prompted the present study on whether these
fungal spores occur in floral and honeydew honeys from different geographical
regions.
Honeydew is an extract from piercing and plant-sucking insects
(Rinchota: Homoptera, e.g. Cinara cofinis Koch, C. pectinatae Nordlinger, C.
pilicornis Hartig, Mindarus abietinus Koch and Physokermes piceae Schrank),
which suck phloem sap, which is rich in nutrients, especially amino acids. To
satisfy their protein needs, these insects need large amounts of sap, which
contains only 1-2% of proteins, though it is high in water content and sugars.
In Italy honeydew is produced between July and September (Persano Oddo et
al., 2000). When production is high on forest trees, honeydew drops fall to the
ground. When the volume of honeydew on the leaves reaches a certain level, it
is collected by honeybees (Apis mellifera L.). Honeybees then transport it to
hives and process it into honeydew honey. Honeydew honey, often called
104

Fungal Diversity
"forest honey", is commercially valuable. Those from silver-fir, oak-trees,
wheat, citrus, etc. are marketed worldwide (Ricciardelli D'Albore, 1998).
Some algae and microscopic fungi, especially sooty moulds, develop in
honeydew (Hughes, 1976). These fungi can thus be traced in the honeydew
sediment (Ricciardelli D'Albore, 1998). In spite of such an interesting fungal
spore content, little mycological analysis has been reported. In routine
melissopalynological analyses fungal structures are only categorized as
"spores" and "hyphae" (Fehlmann, 1911; Gontarski, 1951). Louveaux et al.
(1978) mentioned, that honeydew elements (HDE) consisted of fungal spores
and hyphae of sooty moulds. Especially, the forest honeys from Pinus brutia L.
contain a great number of hyphae and spores (Ricciardelli D'Albore, 1998).
The only study aimed at identifying the spores was carried out by Pérez-Atanes
et al. (2001). A number of asco- and basidiospores together with conidia of
some common hyphomycetes were recorded, but scoleco- and stauroconidia
were not mentioned.
The main objectives of this study were: (a) to further explore the
occurrence of fungal spores in floral and honeydew honeys with special
attention to stauro- and scolecoconidia; (b) to determine if hyphomycete
conidia which are known from aquatic habitats occur in honeys.
Materials and methods
We examined 19 floral honeys from Acacia sp., Castanea sativa, Citrus
sp., Eucalyptus sp., Helianthus annuus, Rhododendron sp., Rosmarinus
officinalis, Rubus sp., Taraxacum officinale and Tilia spp., one polyfloral
honey and 25 honeydew honeys (Table 1). Some floral honey samples (nos. 14,
15, 17) were analysed in Spain by one of us (S-C) and the remainder were
obtained from the National Institute for Apiculture, Bologna and analysed in
Hungary.
Preparation of samples: 10 g were taken from 500 g of previously
homogenised honey, dissolved in 20 ml of distilled water at 40o, centrifuged
for 5 s at 2,500 rpm and allowed to settle. The sediment was recovered in 10
ml of distilled water and again centrifuged. The sediment was then collected
with a Pasteur pipette and dried onto microscope slides at 40o. It was then
mounted in glycerine-gelatine and covered (Louveaux et al., 1978). The entire
surface of each preparation was scanned under phase contrast and fungal
propagules were identified and counted. The detailed melyssopalynological
description of the honeydew honey samples is given in Persano Oddo et al.
(2000).
105

Table 1. Types of honeys examined for fungal spores.
Substratum Source of nectar Pollinator Locality Honey
ref. no.
Floral Honey Castanea sativa Miller. Apis mellifera Italy (North) 1
Floral Honey Unknown Apis mellifera South Africa 2
Floral Honey Unknown Apis mellifera South Africa 3
Floral Honey Unknown Apis mellifera South Africa 4
Floral Honey Unknown Apis mellifera Africa 5
Floral Honey Unknown Apis mellifera New Zealand Pohutukawa 6
Floral Honey Taraxacum officinale
Weber
Apis mellifera Italy Piemonte 7
Floral Honey Acacia sp. Apis mellifera South Africa 8
Floral Honey Citrus sp. Apis mellifera Italy Sicilia 9
Floral Honey Helianthus annuus L. Apis mellifera Italy (Middle) 10
Floral Honey Unknown Apis mellifera Tanzania 11
Floral Honey Rosmarinus officinalis L. Apis mellifera Portugal 12
Floral Honey Unknown Apis mellifera Portugal 13
Floral Honey Eucalyptus sp. Apis mellifera Coastal areas of NW Spain 14
Floral Honey polyfloral Apis mellifera Spain (NW) 15
Floral Honey Rhododendron sp. Apis mellifera Italian Alp 16
Floral Honey Rubus sp. Apis mellifera NW Spain 17
Floral Honey Tilia sp. Apis mellifera Italy (North) 18
Floral Honey Unknown Apis mellifera Mexico 19
Honeydew H Abies alba Mill.+ Picea
excelsa Link
Apis mellifera Italy Tusco-Emilian
Appenines
20
Honeydew H Unknown Apis mellifera Croatia 21
Honeydew H Unknown Metcalfa pruinosa Italy Liguria 22
Honeydew H Unknown Apis mellifera Italy Liguria 23
Honeydew H Abies alba Apis mellifera Greece 24
Honeydew H Unknown Apis mellifera Italy Trentino Alto Adige 25
Honeydew H Unknown Apis mellifera Italy Liguria 26
Honeydew H Abies alba Apis mellifera Greece 27
Honeydew H Unknown Apis mellifera Greece 28
Honeydew H Abies alba Apis mellifera Greece 29
Honeydew H Pinus sp. Apis mellifera Greece 30
Honeydew H Unknown Apis mellifera Italy Lombardia 31
Honeydew H Unknown Apis mellifera Italy Lazio 32
Honeydew H Unknown Apis mellifera Italy Friuli Venezia Giulia 33
Honeydew H Unknown Apis mellifera Italy Abruzzo 34
Honeydew H Unknown Apis mellifera Italy 35
Honeydew H Abies alba Apis mellifera Italy 36
Honeydew H Abies alba Apis mellifera Italy 37
Honeydew H Unknown Apis mellifera Italy Piemonte 38
106

Fungal Diversity
Table 1 continued. Types of honeys examined for fungal spores.
Substratum Source of nectar Pollinator Locality Honey ref. no.
Honeydew H Abies alba Apis mellifera Greece 39
Honeydew H 'forest-type' Apis mellifera Italy 40
Honeydew H Abies alba Apis mellifera Greece 41
Honeydew H Unknown Metcalfa pruinosa Italy 42
Honeydew H Unknown Apis mellifera Italy Liguria 43
Honeydew H Unknown Metcalfa pruinosa Italy Liguria 44
Results and discussion
The species encountered and the numbers of spores in the honey samples
are listed in Tables 2 and 3. Altogether 9 species were found in the floral honey
(Table 2) and 35 in the honeydew honey samples (Table 3). The number of
fungal species ranged from 0 to 3 in the floral honey samples and from 1 to 20
in the honeydew honey samples. The conidial concentration was highest in the
honeydew honey of Abies alba and Picea excelsa (No. 20).
This study focused on the hyphomycete species with stauro- and
scolecoconidia that had been previously seen in rainwater from trees. Over
30% of the floral honey samples and 100% of those from honeydew honey
contained stauro- and scolecoconidia. Common hyphomycetes (e.g. Alternaria,
Botrytis, Cladosporium, Epicoccum, Stemphylium etc.), widely distributed in
the phyllosphere and in many other microhabitats, were omitted. Likewise, it
was not the aim here to extend analyses to yeasts, although they were present
in many samples.
Metschnikowia reukaufii (Fig. 1), a well-known nectar- and flower-
inhabiting yeast, isolated frequently from nectars of different plants (Grüsz,
1927; Eisikowitch et al., 1990), was an exception because it was frequent
mainly in floral honeys. Metschnikowia reukaufii was found in 68% of floral
honeys and 39% of honeydew honeys, but 70% of the total number of its cell
formations was counted in floral honeys. Although M. reukaufii proved to be
frequent and generally distributed in floral honeys, it was absent in all those
from South-Africa. The majority of the yeast cells were seen as "trident",
"aeroplane" or "cross" formations, as commonly found in floral nectars (Pitt
and Miller, 1968). The numbers of yeast cells in floral honeys varied greatly:
an Italian and a Spanish sample had more than 400 cell formations per sample.
The only floral honey sample from Mexico contained this yeast at a very high
concentration, (615 cell colonies/pseudomycelia per sample). Metschnikowia
reukaufii occurred rarely in honeydew honeys, however, in one sample (no. 33)
an unexpectedly high number of cell formations was found, but their source
107

Fig. 1. Cell formations of Metschnikowia reukaufii as frequently seen in floral honeys. Bar =
50 µm.
remained unknown. The occurrence of stauro- and scolecoconidia indicates
that floral honey contains some honeydew. Conversely, the presence of
Metschnikowia reukaufii in honeydew honey may be of floral origin. The
contamination of floral honeys with HDE occurs rarely, because the nectar
harvesting period is earlier than that of honeydew (Sabatini et al., 2000).
Very few other fungal species occurred with very low conidial numbers
in floral honeys (Table 2). These included a single conidium of Diplocladiella
scalaroides (Fig. 19) and conidia probably belonging to species of
Geniculospora (Fig. 26) and Lemonniera (Fig. 24); as well as some conidia of
Tetraploa aristata and Tripospermum spp. (Figs. 3-6) and two unknown
staurospores (Figs. 33-34) were also found.
108

Fungal Diversity
Table 2. Numbers of fungal spores found in floral honey samples. (Honey ref. No. see in Table
1., locality: A=Africa, I= Italy, M=Mexico, P=Portugal, S= Spain, Z= New Zealand.)
Honey ref. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Locality I A A A A Z I A I I A P P S S I S I M
Diplocladiella scalaroides
Arnaud ex Matsush.
- - - - - - - 1 - - - - - - - - - - -
?Geniculospora sp. - - - - - - - - - - - - - - - - - - 1
Lemonniera sp. (?) - - - - - - - - - - - - - - - - - - 1
Metschnikowia reukaufii Pitt
& Miller
15 - - - - 4 - - 53 1 4 195 10 461 233 417 23 14 615
Tetraploa aristata Berk. & Br. - - - 3 - - - - - - - - - - - - - - -
Tripospermum myrti (Lind)
Hughes
- 1 - - - - - - - - - - - - - - - - -
Tripospermum spp. 3 - - - - - 3 - - - - - - - - - - - -
Stauroconidia type 2 - - - 1 - - - - - - - - - - - - - - -
Stauroconidia type 3 - - - - - - - 1 - - - - - - - - - - -
Number of species 2 1 0 2 0 1 1 2 1 1 1 1 1 1 1 1 1 1 3
By contrast, relatively high numbers of scoleco- and stauroconidia of
various species were observed in honeydew honeys (Figs. 2-40). A rich variety
of conidia of Tripospermum species dominated. Conidia were observed in
various stages of development and colony fragments with developing conidia
were sometimes also seen (Figs. 8-9). Conidia of T. camelopardus and T. myrti
could be distinguished (Figs. 2 and 7). Conidia with very long branches (Fig.
4) exceeded the dimensions given for those of T. camelopardus. Many others
could not be identified to species (Figs. 3-6 and 9) due to the great variability
of form, which is a property of species in Tripospermum. Tubaki et al. (1985),
for example, also concluded "that species of the genus are separated only after
careful cultural observations".
Beside Tripospermum species, some 30 other forms, mostly stauro- and
scolecoconidia, occurred in honeydew honeys from Italy and Greece and in the
sole sample from Croatia. Relatively high species numbers occurred in some
Greek (9-11 species) and even higher (8-20 species) in some Italian honeydew
honeys. Conidial numbers in the majority of species were generally low (1-10
per sample). These numbers were, however, higher for other hyphomycete
species. For example, 35 conidia of Ceratosporium cornutum (Fig. 14) and 32
conidia of Retiarius bovicornutus (Fig. 10) were counted in the samples of
honeydew honey from Abies alba and Picea excelsa from the Tusco-Emilian
Appenines, Italy. Matsushima (1975) described Ceratosporium cornutum from
decaying plant material in a terrestrial habitat in Japan and he later collected
this species on decaying wood from Alabama, USA (Matsushima, 1981). Ando
and Tubaki (1984a) found its conidia in rainwater from Zelkova serrata in
109

Figs. 2-9. Tripospermum species. 2. Tripospermum camelopardus. 3-6. Tripospermum spp. 7.
Tripospermum myrti. 8. Part of a Tripospermum colony with conidium initials (arrows:
developing conidia). 9. detached immature conidia of Tripospermum spp. frequently seen in
honeydew honeys. Bar = 50 µm.
110

Fungal Diversity
Table 3. Numbers of fungal spores identified in honeydew honey samples. (Honey ref. No. see
in Table 1., locality: C=Croatia, G=Greece, I= Italy)
Honey ref. No. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Locality I C I I G I I G G G G I I I I I I I I G I G I I I
?Articulospora sp. - - - - - - - - 1 - - - - 1 - - - - - - - - - - -
Atichia sp. 70 - - - 6 - - 21 18 1 - - - - - - - - - 5 - 7 - - -
Camposporium sp. - - - - - - - - - - - - - 1 - - - - - - - - - - -
Ceratosporium
cornutum Matsush.
35 - - - - 5 - - - - - - - 2 - - - - - - - - 1 - -
?Curucispora sp. - - - - - 2 - - - - - - - - - - - - - - - - - - -
Dicranidion sp. - 1 1 1 - 2 - 1 - - - - - - - - - - - - - - - 1 -
Diplocladiella
scalaroides Arnaud ex
Matsush.
- - - - - - - - - - - - - 1 - - - - - - - - - - -
Dwayaangam dichotoma
Nawawi
1 - - - - - - - - - - - - - - - - - - - - - - - -
Dwayaangam sp. - - - - - 8 - - - - - - - - - - - - - - - - - - -
?Geniculospora sp. - - - - - 8 - - - - - - - 1 - - - - - - - 1 - - -
Gyoerffyella ?
myrmecophagiformis
Melnik & Dudka
- 2 - - - - - - - - - - - - - - - - - - - - - - -
Helicosporium sp. - 1 - - - 2 - 1 - - - - - 2 - - - - - - - 1 - - 1
Isthmotricladia sp. - - - - - 2 1 - - - - - - - - - - - - - - - - - 1
?Lemonniera sp. 1 - - - - 1 - 1 1 - - - - - - - - - - - - 1 - - -
Metschnikowia reukaufii
Pitt & Miller
- - - - 1 3 - - 3 2 - - 2 891 - - - 1 - - - 1 5 - -
Mycocentrospora sp. - - - - - - - - - - - - 1 - - - - - - - - - - - -
Oncopodiella sp. - - - - - - - - - - - 2 - - 1 - - - - - - - - - -
Retiarius bovicornutus
D.L. Olivier
32 - - - 1 6 - - - - - - - 6 - 1 - 1 - - - 1 - - -
Tricellula sp. - - - - - - - - - - - - - - - - - 1 - 1 - - - - -
?Tricladium sp. - - - - - - - - - - - - - - - - - - - - - 1 - - -
Trifurcospora sp. 2 - - - - 1 - - - - - - - - - - - - - - - - - - -
Trinacrium
?parvisporum Matsush.
- - - - - 3 - 2 1 - - - - - - - - - - - - - - - -
Trinacrium robustum
Tzean & Chen
2 - - - - - - - - - - - - - - - - - - - - - - - -
Trinacrium subtile Riess - - - - - 1 1 - - - - - - - - - - - - - - - - - -
Trinacrium sp. 8 - - - - 13 - - - - - - 1 1 - - - - - 1 - - - - -
Tripospermum
camelopardus Ingold,
Dann & McDougall
5 - - - - 2 - - - - - - 11 - - - 1 2 - - - - 4 1 -
Tripospermum myrti
(Lind) Hughes
- 31 14 8 - 47 4 - - - - - 118 6 1 8 17 5 66 1 23 1 14 2 3
Tripospermum spp. 191 9 9 5 3 54 3 7 5 - 1 2 32 5 - 12 7 11 8 2 6 - 10 5 4
?Varicosporium sp. 5 - - - 2 - - 1 - - - - 1 1 - - - - - - - - - - -
Unknown helicoconidia - - - - - - - - - - - - - - - - - - - - - 1 - - -
111

Table 3 continued. Numbers of fungal spores identified in honeydew honey samples.
Honey ref. No. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Locality I C I I G I I G G G G I I I I I I I I G I G I I I
Scolecoconidia type 1 551 2 2 - 8 89 - 61 91 10 - - - 6 - - - 11 - 14 6 27 22 - -
Scolecoconidia type 2 - - - - 1 - - 2 - 1 - - - - - - - - - - - - - - -
Scolecoconidia type 3 - - - - - 8 - 4 6 - - - - 2 - - - - - 1 - 4 - - -
Stauroconidia type 1 - - - - - - - - - - - - 2 - - - - - - - - - - - -
Stauroconidia type 4 - - - - - 4 - 1 1 - - - - - - - - - - - - - - - -
Number of species 12 6 4 3 7 20 4 11 9 4 1 2 8 14 2 3 3 7 2 7 3 11 6 4 4
Japan. Retiarius bovicornutus was originally described from S. Africa as a
pollen-capturing fungus in the phyllosphere (Olivier, 1978). It was also
isolated from rainwater on Pinus densiflora in Japan by Ando and Tubaki
(1984c), who believe that it may grow on intact leaf surfaces and produce
"conidia under the impetus of fresh water in the form of rain, morning dew or
mist". They considered this fungus to be a member of the "arboreal aquatic
hyphomycetes". Our present findings, i.e. the occurrence of many conidia of
these two species in honeydew honey from coniferous trees, also support their
wide distribution in canopies, and the possibility of getting conidia into aquatic
habitats from trees.
Conidia of three species of Trinacrium, namely T. parvisporum (Fig. 23),
T. robustum (Fig. 20) and T. subtile (Fig. 21) were identified, though with
some doubts. Most other conidia, however, could only be identified to genus
(Fig. 22). In a recent study a great variety and a relatively great abundance of
conidia belonging to different species of Trinacrium occurred in rainwater
from living trees and treeholes in Hungary throughout the year (Gönczöl and
Révay, 2003, 2004). These findings support the theory that most conidia of
Trinacrium spp. found in foam or stream water may be from canopies.
A conidium with characteristic, dichotomous branching was identified as
Dwayaangam dichotoma (Nawawi, 1985) (Fig. 15). Other forms also
resembled Dwayaangam (Figs. 17-18). Some tetraradiate conidia resembled
Lemonniera spp. (Fig. 24). Some conidia of a Trifurcospora (probably T.
irregularis) (Fig. 25) were seen in two samples from Italy. Together with our
findings, increasing evidence suggests that T. irregularis is widely distributed
on living plants, mostly trees. Conidia were also found in rainwater from
Commelina communis and Pinus densiflora in Japan (Ando et al., 1987).
Czeczuga and Orlowska (1999) recorded conidia of this species in rainwater
from Abies, Acer, Fraxinus and Tilia spp. All stemflow samples from beech
(Fagus sylvatica) in a Hungarian forest also contained many conidia of this
species (Gönczöl and Révay, 2004). Stemflow samples collected from different
112

Fungal Diversity
Figs. 10-19. Stauroconidia in honeydew honeys. 10-11. Retiarius bovicornutus. 12. Tricellula
sp. 13. Dicranidion sp. 14. Ceratosporium cornutum. 15. Dwayaangam dichotoma. 16.
Isthmotricladia sp. 17-18. Young and mature conidia of Dwayaangam sp. 19. Diplocladiella
scalaroides. Bar = 50 µm.
113

Figs. 20-31. Stauroconidia in honeydew honeys. 20. Trinacrium robustum. 21. Trinacrium
subtile. 22. Trinacrium spp. 23. Trinacrium parvisporum. 24. Lemonniera spp. 25.
Trifurcospora sp. 26. Geniculospora sp. 27. Stauroconidia type 4. 28. Gyoerffyella cf.
myrmecophagiformis. 29. Curucispora sp. 30. Articulospora sp. 31. Tricladium sp. Bar = 50
µm.
114

Fungal Diversity
Figs. 32-40. Conidia in floral honeys and honeydew honeys. 32. Atichia sp. 33. Stauroconidia
type 2. 34. Stauroconidia type 3. 35. Varicosporium sp. 36. Stauroconidia type 1. 37. Unknown
helicoconidium. 38. Scolecoconidia type 1 (arrows: short basal extensions on some conidia).
39. Scolecoconidia type 3. 40. Scolecoconidia type 2. Bar = 50 µm.
115

Figs. 41-46. Hypothetical process of accumulation of spores of filamentous fungi and of
colonies of Metschnikowia reukaufii in honeydew honey and floral honey. 41. Honeydew
producing aphids in the phyllosphere. 42. Floral nectar-collecting bees. 43. Honeydew-
collecting bees. 44. Mixing of honeydew and floral nectar. 45. Ingoldian-like fungi in the
phyllosphere. 46. Nectar-inhabiting Metschnikowia reukaufii.
116

Fungal Diversity
tree species in the Black Forest region, Germany, likewise contained conidia of
T. irregularis (Gönczöl, unpubl. obs.).
Unidentified scolecospores (type 1, Fig. 38) were found in unusually
high numbers in two honeydew honey samples from Italy and in one from
Greece. All were poorly resolved under the microscope, due to unsuitable
techniques or mounting medium. Most of them probably belonged to several
hyphomycete species. Basal extensions like those on Filosporella and
Anguillospora were clearly seen on some. Others were seen as broken in the
middle or with a central constriction. Their concentration was especially high
(551 conidia) in the sample from the Tusco-Emilian Appenines. This finding is
interesting because most of the studies on these "canopy fungi" have almost
exclusively reported staurospores.
Various conidia of Atichia sp. (Fig. 32) were encountered in an Italian
honeydew sample and five in a Greek one. Atichia spp. have been reported as
epiphytic fungi on various trees and shrubs in both tropical and temperate
regions (Meeker, 1975). However, a search in the literature suggests that "the
members of Atichiaceae are true saprophytes living on honeydew like the
members of the Capnodiaceae and that they therefore should be included in the
sooty moulds group" (Fraser, 1936). The high conidial concentration in
honeydew honey from Picea excelsa and Abies alba in the Italian sample and
occurrence of conidia in almost all Greek honeydew honeys from Abies alba
also support the above hypothesis.
We also observed some microorganisms other than fungi. Some
individuals of Vorticella spp. in the floral honeys of Castanea, Citrus,
Eucalyptus, Rhododendron and Tilia and Vorticella, Euglena, Paramecium and
diatoms (e.g. Cyclotella, Cymbella, Merismopedia and Nitzschia spp.) in
honeydew honeys were often seen. Whether the occurrence of these
microorganisms reflects the existence of wet or aquatic microhabitats in
canopies where some of the aquatic hyphomycetes may find adequate
conditions for growth and sporulation requires more studies.
It is well known that honeydew serves as a nutrient for the sooty moulds
(e.g. metacapnodiaceous fungi) (Reynolds, 1975; Hughes, 1976). However, we
believe that honeydew acts only as a trap for the hyphomycete conidia found in
the present study and it is not known if these can utilize honeydew as sooty
moulds do. Due to the collecting activities of insects (primarily honeybees) the
spores trapped in the honeydew will therefore accumulate in honeydew honey.
This process is proposed in Figs. 41-46. We believe that honeydew, as with
rainwater, merely washes spores (and sometimes other fungal elements, see
Fig. 8) off tree surfaces. Rainwater possibly due to its much higher volumes
and displacement, gathers fungal spores on aerial plant surfaces more
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efficiently than honeydew. On the other hand honeydew honeys can preserve
fungal spores (probably in inactive state) for a long time. Therefore honeydew
honeys bear important mycological information on the source region.
One of the most intriguing questions is where do these fungi with stauro-
and scolecoconidia, occurring regularly in rainwater and honeydew honey, live
on trees. Living trees offer various habitats for saprotrophs, endo- or epibionts
and parasitic fungi where they can find adequate conditions for short or long-
term existence. Consequently rainwater or any other liquid (e.g. honeydew) on
plant surfaces may contain a great variety of spore assemblages. However, we
have little evidence of the active presence of “aquatic hyphomycete” species in
canopies.
Numerous attempts have been made to find an appropriate term (e.g.
arboreal aquatic hyphomycetes, canopy fungi, terrestrial aquatic
hyphomycetes, terrestrial ingoldian hyphomycetes, terrestrial aquatic fungi) for
this fungal group. To create a really adequate term we need to culture them, to
establish their correct taxonomic position and to study their growth habits in
nature. The same is true for traditional aquatic hyphomycetes: more data are
needed to confirm whether they are actually able to exist on aerial, intact or
dead parts of canopies. Experiments are also needed to better understand their
physiological relationship with free water and possibly with some other
environmental factors.
Acknowledgements
The authors are grateful to Dr. Enrique Descals (Inst. Mediterr. de Estudios Avanzados
de las Baleares, CSIC-UIB) for his comments, discussion during manuscript preparation and
language corrections. The authors thank to Gábor Péter (University of Horticulture and Food
Industry, Hungary) the identification of Metschnikowia reukaufii and to Krisztina Buczkó
(Hungarian Natural History Museum) the identification of diatoms.
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