Warsaw University of Life Sciences, Warsaw, Poland
LIGHT AND SCANNING MICROSCOPE OBSERVATIONS
OF CLADOSPORIUM UREDINICOLA GROWTH
ON RUST FUNGI
T.M. Dolińska, A. Bartkowska and M. Schollenberger
Abstract
In the years 2009–2010 relationship between the hyperparasite Cladosporium
uredinicola and various rust fungi was investigated. Fourteen various rusts were
tested under stereomicroscope, light and scanning microscope in order to confirm
the relations between the fungi. The hyperparasite produced olive green mycelium
on 10 species of rust fungi. Gymnosporangium spp. and Tranzschelia spp. were infected most frequently. The type of the sori also proved important – the most intensive infection occurred on the spermogonia and aecia. Directly close to the host
spore surface, C. uredinicola produced infection cushions as well as structures similar to appressorium. The hyperparasite often caused spore deformation leading to
their destruction. In some cases the hyphae of the fungus plunged into the rust
spore surface, which may confirm the fact that it produced lytic enzymes.
Key words: Cladosporium uredinicola, hyperparasitism, fungicolous fungi, rusts
Introduction
The genus Cladosporium is one of the most common and worldwide spread
among fungi (Dugan et al. 2004). For the first time it was described by Link in
1816. At present this genus includes over 770 species which have various strategies of obtaining nutrients (Dugan et al. 2004). There are many saprotrophs
among Cladosporium spp., which decompose dead organic matter thus obtaining
nutrients which are necessary for them to live (Ellis 1971). Many species are
epiphytes growing on plants or endophytes occurring in the leaf tissues (Fisher
and Petrini 1992, El-Morsy 2000).
Phytopathologia 61: 37–44
© The Polish Phytopathological Society, Poznań 2011
ISSN 2081-1756
38
T.M. Dolińska, A. Bartkowska and M. Schollenberger
Pathogens infecting fruits of such plants as cucumber, tomato or water melon
are also noted within that genus (Fajola 1979, Narain et al. 1985, Batta 2004).
Cladosporium spp. may also cause diseases in human beings, e.g. allergic lung
mycoses or keratomycoses (de Hoog et al. 2000, Chew et al. 2009). In the natural
environment there are also such species of Cladosporium, which obtain nutrients
from other fungi. That phenomenon is described as hyperparasitism and it is often
observed in members of that genus (Ellis 1971). An excellent example is the
necrotroph Cladosporium uredinicola noted on uredinial sori and teliosori of rust
fungi (Spegazzini 1912, Jeffries and Young 1994). The fungus produces conidiophores and conidia on plant parasites such as Puccinia recondita and Cronartium
quercuum (Morgan-Jones and McKemy 1990, Sheroze et al. 2002). Srivastava et al.
(1985) claimed that the hyperparasite infected about 28% of the teliosori surface
of the rust Puccinia horiana occurring on the leaves of Chrysanthemum morifolium and
44% of the teliosori surface of Puccinia arenariae on Dianthus barbatus. The authors
also observed less extensive infections of the rust teliosori of P. malvacearum on
Althaea rosea and P. glomerata on the Senecio jacobaea leaves. The mycelium of C.
uredinicola was also found on P. cestri, P. henningsii, P. violae (Spegazzini 1912, Ellis
1976, Traquair et al. 1984). Barros et al. (1999) confirmed the occurrence of that
hyperparasite on the aeciospores of Puccinia puta on the leaves of Ipomoea fistulosa.
Apart from the mentioned rusts, C. uredinicola may also infect fungi from the
Erysiphales order, and many species of insects such as whiteflies or aphids
(Abdel-baky et al. 1998, Dugan and Glawe 2006). In all the investigations the
hyperparasite has never infected plant tissues.
In Poland at present there are no research works concerning the hyperparasites
of the Cladosporium genus and its possible use for biological protection of plants
against diseases. The aim of the present research was to study the range of the
hyperparasite hosts and to determine the relations between C. uredinicola and its
hosts.
Material and methods
Cladosporium uredinicola used in this study was isolated from the rust Tranzschelia
pruni-spinosae collected in August 2008 in the area of the Skarpa Ursynowska Reserve
in Warsaw (52°9'56'' N, 21°3'0'' E). The hyperparasite was cultured under laboratory
conditions on the potato dextrose agar (PDA, Difco) at 20°C. The species was identified with Ellis’ key (1976). Samples of plants infected by the rusts of the Melampsora,
Puccinia, Cumminsiella, Gymnosporangium, Phragmidium, Cronartium and Tranzschelia
genera were collected from the Skarpa Ursynowska Reserve in Warsaw and from the
experimental field of the Department of Plant Pathology (Warsaw University of Life
Sciences) in Warsaw (52°9'49.68'' N, 21°2'52.87'' E). The fungi were identified by its
morphology and host plants according to Majewski’s keys (1977, 1979).
Leaves with symptoms of the rust were cut into 15 × 15 mm pieces and disinfected with 2% sodium hypochloride. Then they were placed on the wetted blot-
Light and scanning microscope observations of Cladosporium uredinicola....
39
ting paper in Petri dishes and inoculated with the suspension of C. uredinicola
spores. The inoculum was obtained by washing off spores from seven-day-old cultures growing on PDA with sterile, distilled water with few drops of Tween 20
(Difco). The spore solution was filtered in order to remove the excess of mycelium
and then, using the hemocytometer, its concentration was determined. Artificial
inoculation was performed by applying 1 ml of spores suspension at a concentration of 5 × 107 conidia per 1 ml. For control, leaves with the rust symptoms were
treated with sterile water. To protect leaves against the loss of moisture, Petri
dishes were sealed with Parafilm. The dishes were kept under laboratory conditions at 20°C. The experiment was performed in 10 replications. After 72 h of incubation leaves were examined under a stereomicroscope Olympus SZ11. The
results were determined using the following criteria:
0 – no parasitism or less than 10%,
1 – 10–20% of rust sori were overgrown by the parasite,
2 – 20–50% of rust sori were overgrown by the parasite,
3 – 50–100% of rust sori were overgrown by the parasite.
Then, the tested leaves were dried, cut into small fragments and covered with
gold and examined using a scanning microscope FEI Quanta 200.
The observations in the hanging drop using a light microscope were also carried
out. For that examination aeciospores of Tranzschelia pruni-spinosae and Gymnosporangium sabinae were put in a drop of water with conidia of C. uredinicola. The slides
were protected against the loss of moisture using vaseline. Additionally they were
placed in Petri dishes with wetted blotting paper and sealed with Parafilm. After 72
h results were observed using a light microscope.
Results
The hyperparasite attacked 10 out of 14 tested fungi. Rust sori treated with the
suspension of C. uredinicola spores were overgrown by the fungus hyphae. Already
two–three days after inoculation the hyperparasite produced conidiophores and
conidia on the surface of the sori. The intensity of infection varied depending on
the type of sori and species of the rust. Mostly the infection was observed on aecias
– the mycelium of C. uredinicola overgrew over 90% of their surface (Phot. 1). The
surface of uredinial sori was covered by the mycelium of the hyperparasite in
20–50% (Phot. 2). Teliosori were infected very rarely, only 10% of their surface
were colonized by structures of the hyperparasite. The control represented by the
rust fungi treated with water remained healthy. No relations between the plant tissue and hyperparasite were observed. Cladosporium uredinicola formed a close relation mainly with the genera Gymnosporangium and Tranzschelia. Some species of
Puccinia and Phragmidium were also attacked. The remaining rusts were not infected
with hyperparasite (Table 1).
Light microscope was useful for showing the growth of C. uredinicola towards
the aeciospores of T. pruni-spinosae and G. sabinae in the hanging drop. A callosity
40
T.M. Dolińska, A. Bartkowska and M. Schollenberger
Phot. 1. Aecia of Gymnosporangium sabinae
completely overgrown by Cladosporium
uredinicola (photo by T.M. Dolińska)
Phot. 2. Uredinia of Puccinia graminis with
conidiophores of Cladosporium uredinicola
(photo by T.M. Dolińska)
similar to appressorium which adhered to the spore surface was formed at the ends
of the hyperparasite hyphae (Phot. 3). Moreover, the fungus produced long hyphae
which coiled round the aeciospores (Phot. 4). Spore cells sank and changed their
initial shape. Sometimes a breaking of the cell wall and total destruction of cells
were observed.
Observations under the scanning microscope confirmed that conidiophores
and conidia of C. uredinicola developed on the surface of spermogonia and aecia of
both G. sabinae and T. pruni-spinosae (Phots. 5 and 6). The host sori were usually infected by many hyphae of the hyperparasite which often destroyed its surface. At
Table 1
Results of Cladosporium uredinicola infection in selected rust fungi (scale 0–3)
Host plant
Species of rust fungi
Impatiens parviflora
Puccinia komarovii
Malva sylvestris
Puccinia malvacearum
Triticum aestivum
Puccinia graminis
Populus alba
Melampsora populnea
Bellis perennis
Artemisia campestris
Secale cereale
Salix alba
Puccinia lagenophorae
Puccinia tanaceti
Puccinia recondita
Melampsora salicis-albae
Mahonia aquifolium
Cumminsiella mirabilissima
Pyrus communis
Gymnosporangium sabinae
Ribes nigrum
Cronartium flaccidum
Sorbus aucuparia
Potentilla anserina
spermogonia
–
–
–
–
Inoculated rust sori
aecia
uredinia
1
–
–
–
–
–
–
–
Gymnosporangium cornutum
3
3
Phragmidium potentillae
–
–
Anemone ranunculoides Tranzschelia pruni-spinosae
3
–
3
0
0
–
2
–
3
–
3
–
2
–
–
2
0
1
2
telia
–
–
–
–
–
0
1
–
–
0
–
–
–
–
–
–
1
–
–
–
0
Light and scanning microscope observations of Cladosporium uredinicola...
41
Phot. 3. A callosity similar to appressorium at
the ends of hyperparasites hyphae on the
surface of an aeciospore of Tranzschelia
pruni-spinosae (photo by T.M. Dolińska)
Phot. 4. Cladosporium uredinicola hyphae coiled
around aeciospores of Tranzschelia pruni-spinosae
(photo by T.M. Dolińska)
Phot. 5. Conidiophore of Cladosporium uredinicola
grown from aeciospore of Gymnosporangium
sabinae (photo by T.M. Dolińska)
Phot. 6. Aeciospore of Tranzschelia pruni-spinosae
with hyperparasite (photo by T.M. Dolińska)
the site of the direct contact between the fungi either appressorium or branched infection cushions were formed. Sometimes the hyperparasite penetrated the surface
of the rust spore which confirms its use of various lytic enzymes.
Discussion
The present research demonstrates the relation between C. uredinicola and various species of the rust fungi. A visible symptom of infection is olive green mycelium of C. uredinicola which grows on the sori surface. It was confirmed by the
observations under the stereoscopic microscope. Under laboratory conditions 10
genera of the rust proved to be the hosts for the hyperparasite. The investigations
show that the hyperparasite often infects rusts of the Puccinia genus (Table 1). Sim-
42
T.M. Dolińska, A. Bartkowska and M. Schollenberger
ilar observations were reported by Ellis (1976), Srivastava et al. (1985) and Barros
et al. (1999). Morgan-Jones and McKemy (1990) demonstrated that C. uredinicola is
the hyperparasite for the rust of the Cronartium genus. In our research the Cronartium flaccidum causing rust of Ribes nigrum was not infected. Also the Melampsora and
Cumminsiella genera were not attacked. Three of the tested rusts were totally overgrown by the hyperparasite mycelium – G. sabinae, G. cornutum and T. pruni-spinosae.
Both the type of sori and the host species seem to be important. Spermatia and
aecia were most often infected by the hyperparasite. The hyperparasite also infected the uredinial sori, however, contrary to Srivastava’s et al. (1985) report, the
telio stage of the rust fungus was infected very rarely if at all.
The observations under the light microscope showed that the hyperparasite has
the ability to produce long hyphae, which wrap up spores of the rust. Cladosporium
uredinicola also produces other structures similar to appressoria or infection cushions (Tsuneda and Hiratsuka 1979, Moricca et al. 2001). Finally the fungus destroys the cell wall of the host and deforms its spores. It may indicate the
participation of lytic enzymes or other toxic chemical compounds in the infection
process (Fukushima et al. 1993, Sakagami et al. 1995, Assante et al. 2004).
The present paper demonstrates that in the future there is possibility of using
C. uredinicola in the biological protection of plants against some rusts but very important is continuing research about relationships between these organisms.
Streszczenie
OBSERWACJE MIKROSKOPOWE WZROSTU CLADOSPORIUM UREDINICOLA
NA GRZYBACH RDZAWNIKOWYCH
W latach 2009 i 2010 przeprowadzono badania nad związkiem nadpasożyta
Cladosporium uredinicola i różnych gatunków grzybów rdzawnikowych. Materiałem
badawczym były liście z objawami rdzy, które inokulowano zawiesiną zarodników.
Obserwacje z wykorzystaniem mikroskopu stereoskopowego wykazały, że oliwkowozielony nalot grzybni nadpasożyta pojawił się na powierzchni 10 z 14 testowanych gatunków rdzy. Grzyby rdzawnikowe rodzajów Gymnosporangium i Tranzschelia
były porażane przez nadpasożyta w największym stopniu – nalot C. uredinicola obserwowano na ponad 90% ich powierzchni. Zwrócono również uwagę na fakt, iż
nadpasożyt najczęściej porażał spermogonia oraz ecja, co może być związane ze
specjalizacją badanego grzyba. Obserwacje z użyciem mikroskopu świetlnego i
skaningowego pozwoliły stwierdzić, że grzyb na powierzchni ścian komórkowych
zarodników gospodarza produkował specjalne rozgałęzione strzępki lub zgrubienia podobne do appressorium. Nadpasożyt często powodował deformacje zarodników rdzy, a także wielokrotnie doprowadzał do ich degradacji. W niektórych
przypadkach strzępki nadpasożyta przenikały do wnętrza zarodników rdzy, co
może być dowodem na wytwarzanie przez nadpasożyta enzymów litycznych.
Light and scanning microscope observations of Cladosporium uredinicola...
43
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44
T.M. Dolińska, A. Bartkowska and M. Schollenberger
Authors’ address:
Tatiana M. Dolińska M.Sc., Dr. Agnieszka Bartkowska, Dr. hab. Małgorzata
Schollenberger, Department of Plant Pathology, Warsaw University of Life
Sciences, ul. Nowoursynowska 159, 02-787 Warszawa, Poland, e-mail:
[email protected]
Accepted for publication: 10.08.2011