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Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
Parasitic fungi on roses
Marcel Pârvu1, Alina E. Pârvu2
1
Department of Biology, Faculty of Biology and Geology, "Babes-Bolyai" University, 42 Republicii Street, 400015 ClujNapoca, Romania.
2
Department of Pathophysiology, Faculty of Medicine, "Iuliu Hatieganu" University of Medicine and Pharmacy, 3 Victor
Babes Street, 400012 Cluj-Napoca, Romania.
Corresponding authors: [email protected], [email protected]
Roses are susceptible to many diseases, and some of the major ones are caused by parasitic fungi like Podosphaera
pannosa, Diplocarpon rosae, Phragmidium mucronatum and Botrytis cinerea.
Powdery mildew is caused by P. pannosa, one of the most important fungal diseases of roses. P. pannosa mycelium and
conidia are common on leaves and shoots of roses and infections are limited to the epidermal cells. Inside the host cell, P.
pannosa haustoria provide a large area of contact with the host. A natural antagonist of P. pannosa is Ampelomyces
quisqualis mycoparasite which forms typically pycnidia within different host structures.
Black spot is caused by D. rosae and rose rust by P. mucronatum, a fungus which in the biological cycle presents five
types of spores. Mycoparastism relations between roses and D. rosae and P. mucronatum were studied on the base of
ultrastructural plant changes produced by the pathogens and on the mycelium development in plant tissues.
Rose gray mold is caused by the B. cinerea species and the disease occurs on leaves or flower buds of plants. B. cinerea
produces abundant gray mycelium and long and branched conidiophores that have ovoid and one-celled conidia. The B.
cinerea conidia had numerous randomly positioned protuberances and a regular cell wall with a two-layer structure. The
fungus frequently produces black and irregular sclerotia with distinct layers. The B. cinerea conidia lost viability due to
severe ultrastructural changes induced by some plant extracts as Chelidonium majus and Berberis vulgaris.
Key words: electron microscopy, fungal, parasitism, sporulation, ultrastructure
1. Introduction
Roses continue to be one of the most popular garden flowers, as well as one of the most economically important
ornamental flowers that are grown in the worldwide. In addition to their ornamental qualities, they possess some
therapeutically important properties, for example the high levels of vitamin C and cancer-preventing compounds present
in rose hips[1]. The susceptibility of roses to disease is the greatest risk for their quality. The major pathogens causing
disease on rose include fungi, bacteria, nematodes, and viruses [2]. Several rose pathogens are capable of serious
damage. Therefore, substantial research targeted the biology of rose pathogens, in order to increase rose’s resistance, to
avoid excessive use of pesticides and to extend the use of biocontrol methods.
The major parasitic fungi on roses are Podosphaera pannosa, Diplocarpon rosae, Phragmidium mucronatum, and
Botrytis cinerea. Significant yield losses due to fungal attack limit both rose productivity and commercial value [3,4].
2. Podosphaera pannosa
Powdery mildew is caused by the fungus Podosphaera pannosa (syn. Sphaerotheca pannosa var. rosae) and it is one of
the most important fungal diseases occurring on roses, both in the garden and in the greenhouse. This disease appears
on roses year after year and causes reduced flower production and weakening of the plants by attacking their buds,
young leaves, and growing tips [5].
P. pannosa are obligate, biotrophic fungi, meaning they can survive only on cells in specific living hosts. Despite
their restrictive host specificity, powdery mildews are ubiquitous [2]. The mycelium and conidia of P. pannosa are
common on leaves and shoots of cultivated and wild roses [6]. On young leaves the disease appears at first as slightly
raised blister-like areas that soon become covered with a grayish white powdery fungus. As the leaves expand, they
become curled and distorted, leading to shriveling and defoliation. On older leaves, as fungus grows, appear large white
patches that cause little distortion but may eventually become necrotic. Young rapidly extending stem tissue can
become infected too, often where a thorn attaches. The infection generally will persist as the stem matures, resulting in
irregular powdery patches of fungus on the stem [2]. Sometimes buds are attacked, become covered with white mildew
before they open, and either fail to open or open improperly [5].
P. pannosa infections are restricted to the epidermal surface. The fungus produces white mycelia that grow on the
surface of the plant tissues and forms short and erect hyphae or conidiophores. At the tip of each conidiophore, chains
of 5 to 10 ellipsoid-ovoid conidia (asexual spores) are produced (Figure 1A). Sexual spores are occasionally produced
in spherical structures, ascomata, which appear as reddish-brown dots in the hyphal mats [5,7]. After germination, an
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appressoria develops at the end of the germination tube, which attaches the mycelium to the plant surface by a fine
slime layer. A penetration peg emerges through a pore in the appressorium and enters the cuticle and underlying
epidermal cell wall. In the epidermal cell, the penetration peg enlarges to form the haustorial neck. From the center of
the attachment of the appressoria, multilobed, globose mature haustoria are formed. Inside the host cell, P. pannosa
haustoria provide a large area of contact with the host (Figure 1B). Haustoria continue to form as hyphae extend along
the leaf surface [2]. The haustoria serve to absorb nutrients for the fungus from the rose host. The absorption of
nutrients from rose cells may sometimes lead to their death and in the affected areas photosynthesis is greatly reduced
[5].
A
B
Fig. 1 Podosphaera pannosa: A. Light microscopic views of conidiophore (a) and conidia (b); B. Transmission electron micrograph
of cross section through leaf rose showing a haustorium (h) and haustorial lobe (hl) of fungus in epidermal cell.
In cold weather the production of conidia ceases and cleistothecia may be formed. Cleistothecia form occasionally
toward the end of the season. Each cleistothecium contains a single ascus with 8 ellipsoid-ovoid spores [7]. Ascospores
and conidia are carried by wind to young green tissues, and if the temperature and the relative humidity are sufficiently
high spores germinate and infect these tissues.
Control of rose powdery mildew relies mostly on the application of a variety of fungicides. However, these
fungicides can be phytotoxic, and could cause the selection of resistant populations of rosae. For these reasons,
alternative control measures like mineral salts, oils, plant extracts or biological control agents, in combination or as a
replacement for fungicides are needed [8,9]. The oldest known and the commonest natural antagonists of powdery
mildews is Ampelomyces quisqualis Ces [4,7,10,11]. The interactions between host plants, powdery mildew fungi and
Ampelomyces mycoparasites are one of the most evident cases of tritrophic relationships in nature. When applied alone,
A. quisqualis provides good control of rose powdery mildew [10].
Conidia of A. quisqualis are produced in pycnidia developed intracellularly in the mycelia of powdery mildew fungi
[6,12,13]. A. quisqualis forms pale golden brown pycnidia which have different shapes (pear-shaped, spindle-shaped,
spherical) depending on the P. pannosa fungus organ (hyphae, conidiophores and cleistothecia) in which they develop
and act as parasites (Figure 2).
When parasitizing conidiophores, A. quisqualis pycnidia are pear-shaped. In the case of parasitism of hyphae they are
spindle-shaped and in the case of parasitism of cleistothecia they are almost spherical. Pear-shaped and spindle-shaped
pycnidia of A. quisqualis are formed first, and spherical pycnidia appear at the end of the mycoparasite development
cycle. The A. quisqualis mycoparasite pycnidiospores are one-celled, hyaline and smooth, with round, straight or
slightly curved ends, and are embedded in a mucilaginous matrix inside the pycnidia [14]. In the presence of water,
these matrices swell to several times their normal diameter, and conidia are released from intracellular pycnidia by the
rupture of the pycnidial wall. Under high humidity conditions conidia germinate and the resulting A. quisqualis hyphae
can penetrate the hyphae of powdery mildews in their vicinity. After penetration, the hyphae of Ampelomyces invade
the P. pannosa mycelia internally, and produce their pycnidia mostly in the conidiophores and young, immature
ascocarps of powdery mildews. Occasionally, they also produce pycnidia in the invaded hyphal cells. The life cycle
starts again when pycnidia are mature [10].
Cross sections of A. quisqualis pycnidia showed that they have different shape and their wall is composed of cells of
different shapes and sizes. The cells of the internal wall of pycnidia contain many lipids and have a comparatively
larger diameter than those situated on the pycnidia surface. In the centre of the pycnidia is the tissue which forms the
conidia (Figure 3).
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A
B
Fig. 2 Ampelomyces quisqualis: light microscopic views of pycnidium shapes (a) and conidium (b); A. Pear-shaped; B. Spindleshaped.
A. quisqualis conidia can be dispersed within the same plant by rain or water run off from plant surfaces. It can also
spread over long distances as hyphal fragments in parasitized and detached powdery mildew conidia. Ampelomyces was
found to produce pycnidia saprophytically in the senescent or dead plant tissues at the end of the season, suggesting that
these structures served as overwintering structures for Ampelomyces in the field. The conidia, the pycnidial cells and the
cells of the resting hyphae of Ampelomyces produced in the mycelia of powdery mildews during the previous season
can all initiate the life cycle of these mycoparasites in the spring [10].
Ampelomyces is good for P. pannosa biocontrol because the appearance of mature cleistothecia is affected by the
hyperparasite, limiting the attack of powdery mildew on roses [14]. On the other hand, A. quisqualis is tolerant to
several fungicides used against powdery mildews, so that integrated control may be possible [15].
A
B
Fig. 3 Transmission electron micrograph of cross section through Ampelomyces quisqualis pycnidia: A. Pycnidium (p) and conidium
(c); B. Details of pycnidium (p) wall and conidium (c) ultrastructure.
3. Diplocarpon rosae
Black spot is caused by D. rosa, a fungus that is obligate to the genus Rosa, and does not infect any other plant taxa. It
is hemibiotrophic, because it is parasitic on living host tissue and also has some ability for saprophytic growth [16]. The
disease appears as black spots on the leaves that may coalesce to produce large, irregular, black lesions. The leaf tissue
around the lesions may turn yellow, and often entire attacked leaves become yellow and fall off prematurely, leaving
the canes almost completely defoliated [5]. Black spot is the most damaging rose disease worldwide [17,18].
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D. rosae is mainly spread through asexual spores, conidia. The sexual stage, represented by ascospores, is extremely
rare and plays no part in the disease cycle. The fungus overwinters as mycelium, ascospores, and conidia in canes and
fallen infected leaves. In the spring conidia are dispersed via water splash. The mycelium grows in the mesophyll and
forms acervuli and conidia at the upper surface. The primary infection of leaves is caused by direct penetration of
conidia and ascospores. Available water is necessary for the fungus to germinate and directly penetrate the epidermis of
rose leaves and stems [5,19].
On susceptible rose genotypes D. rosae fungus produces Marssonina – type of 2-celled hyaline conidia from
acervuli, within infection sites on leaves and stems, between the outer wall and cuticle of the epidermis (Figure 4).
Subcuticular hyphae radiate from the infection site followed by branching intercellular hyphae that give rise to
intracellular haustoria [16]. Conidia push up and rupture the cuticle [5].
A
B
Fig. 4 Diplocarpon rosae: A. Scanning electron micrograph of acervulus (a) and conidia (c) of the fungus; B. Transmission electron
micrograph showing hyphae (hy) between the outer wall and cuticle (c) of the epidermis.
Conidial morphology and colony color are quite variable among isolates when grown in culture, due to their genetic
diversity reflected in pathogenic race diversity [19-21]. D. rosae fungus penetratation in cells and intercellular spaces of
leaf mesophyll causes irreversible ultrastructural changes of the affected cells (Figure 5). The intercellular penetration
damages plant cell membranes and increases nutrient leakage into intercelullar spaces. At the same time the
physiological and biochemical processes of the plant cells are strongly modified by mycoparasitism, affecting the
overall functioning of plant tissues and the growth process [22].
A
B
Fig. 5 Transmission electron micrograph of cross section through leaf rose showing hyphae (hy) of Diplocarpon rosae: A. In the
tissue cells and intercellular spaces; B. In intercellular spaces and penetrating in the mesophyll cell (mc).
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4. Phragmidium mucronatum
Rose rust is caused by the P. mucronatum fungus and this disease appears in spring and persists until the leaves fall.
Susceptibility to rust varies widely among rose cultivars. The rose rust appears on leaves as yellow to red circular spots
on the upper surface, corresponding to pustules of red, orange or black spores on the lower surface. In late summer, on
the lower surface of the leaf there are black pustules which contain teliospores. Rose rust often causes the death of rose
shrubs due to premature defoliation of plant.
P. mucronatum is an obligate parasite and an autoecious and macrocyclic fungus. During its biological cycle it
presents five types of spores (teliospores, basidiospores, pycniospores, aeciospores and uredospores) appearing in a
definite sequence. The urediniospores are one-celled and yellowish-orange (Figure 6), and the teliospores contain 6-8
cells with very dark and rough walls and a long stalk (pedicel) which becomes easily detached from the lesions of
leaves [6] (Figure 7).
A
B
Fig. 6 Scanning electron micrograph of the fungus Phragmidium mucronatum showing: A. Uredospores (u) and teliospores (t) on
lower epidermis; B. Uredospore.
Typically, P. mucronatum fungus produces intercellular, hyaline and septate hyphae and haustoria, all of which are
involved in the absorption of nutrients from living cells of the host plants [23] (Figure 8).
A
B
Fig. 7 Scanning electron micrograph of the fungus Phragmidium mucronatum showing: A. Teliospore with dark and rough wall and
a long stalk; B. Teliopsore with dark and rough wall (detailed).
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A
B
Fig. 8 Transmission electron micrograph of cross section through leaf rose showing the Phragmidium mucronatum fungus: A.
Hyphae (hy) of the fungus in the intercellular spaces and irreversible changes in the mesophyll cells; B. Haustorial lobe (hl) in the
mesophyll cell.
5. Botrytis cinerea
B. cinerea is a necrotrophic opportunistic plant pathogenic fungus, also known as “gray mould fungus”. It causes
serious pre- and postharvest diseases in more than 200 plant species, including agriculturally important crops and
harvested commodities, such as grapes, tomatoes, strawberries, cucumbers, bulb flowers, cut flowers and ornamental
plants [24]. The broad host range of B. cinerea results in great economic losses, not only during growth but also during
storage and transportation of products [25]. Necrotrophs kill their host cells by secreting toxic compounds or lytic
enzymes and also produce an array of pathogenic substances that can subvert host defences [26]. B. cinerea strains are
highly genetically and physiologically variable and several strains developed resistance to most of the fungicides used
to control them [24,27,28].
Rose gray mold occurs on leaves or flower buds of plants. B. cinerea produces abundant gray mycelium and long and
branched conidiophores, that have ovoid and one-celled conidia (Figure 9A). B. cinerea conidia appear dark because of
melanin, which protects the spores against enzyme action and probably UV [29]. The mycelium grows and invades the
tissues, which become covered with a whitish-gray mold. The surfaces of dry B. cinerea conidia and other Botrytis spp.
have many short protuberances (Figure 9B). Hydration and redrying causes the disappearance of these protuberances
[30,31].
A
B
Fig. 9 Botrytis cinerea: A. Light microscopic view of a conidiophore with conidia; B. Transmission electron micrograph of a cross
section showing hyphal cells from the inner layer of sclerotium, embedded in a polysaccharide matrix (CW. cell wall; N. nucleus; C.
cytoplasm; L. lipids; PM. polysaccharide matrix).
The B. cinerea conidium ultrastructure presents a regular cell wall, approximately 300–400 nm thick, with a twolayer structure, plasmalemma, and cytoplasm matrix with nucleus, mitochondria and vacuoles. The cell wall external
layer is thin and electron dense and the inner one is thick, uniform and less electron dense. The plasmalemma tightly
adhered to the cell wall. The cytoplasm matrix (cytosol) is uniformly distributed, and the nucleus is up to 2 µm in
diameter and ovoid or spherical in shape. Among cell organelles, mitochondria are numerous, usually ovoid and
medium electron dense. Vacuoles are similar in size to mitochondria [30,32].
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The B. cinerea fungus frequently produces black and irregular sclerotia with distinct layers at the surface of infected
tissues and the fungus overwinters in this form. Transverse sections of B. cinerea sclerotia showed a cortex more
compact than the medulla, with less extracellular matrix between the cells. The rind cells had darkly pigmented septa.
The medullary cells were embedded in a continuous polysaccharide extracellular matrix, uninterrupted by lacunae
showed in Figure 9B.
The morpho-functional integrity of fungal cell components is required in order to maintain their viability and
germination capacity. It has been demonstrated that Chelidonium majus [33] and Berberis vulgaris [34] plant extracts
induced important irreversible ultrastructural changes to B. cinerea conidia which were visualized by electron
microscopy (TEM) [35].
Important antifungal activity of Berberis spp. has been demonstrated against some fungal strains with hydroalcoholic
extracts, aqueous extract, methanolic or crude extracts, and alkaloidal fractions [36]. Alcoholic extracts provide more
complete extraction and include fewer polar compounds [37]. The in vitro antifungal activity of berberine isolated from
the same sources has also been investigated and it was found that berberine alkaloids are cationic antimicrobials.
Twenty-two alkaloids of medicinal importance have been reported so far from the roots, stems, leaves and fruit of
Berberis spp. The alkaloid content differs in Berberis from different areas, species and organs [38].
Examination by SEM revealed that B. vulgaris bark extract, at its MIC, induced large-scale damage to B. cinerea
conidia, because the surface protuberances from the control disappeared. On TEM micrographs, B. vulgaris bark extract
caused a disruption of the B. cinerea conidial cell wall, the external layer was more electron dense, the plasmalemma
and the cytoplasm of the B. cinerea conidia had shrunk and detached itself altogether from the cell wall, the organelles
and nucleus were also partly destroyed. Berberine treatment caused similar changes to the B. cinerea conidia as B.
vulgaris bark extract [34].
C. majus is a common, poisonous herbaceous perennial from the poppy family, commonly known as celandine. Plant
extracts and their purified compounds have antibacterial, antiviral and fungicidal effects both in vitro and in vivo. Their
properties were attributed mainly to alkaloids, several flavonoids and phenolic acids. The main alkaloids from C. majus
extracts are chelidonine, chelerythrine, sanguinarine, coptisine and berberine [33,39].
On the SEM micrographs of the B. cinerea conidia treated with MFC of C. majus extract, the shape and size did not
change but the surface protuberances disappeared. The TEM micrographs showed important irreversible ultrastructural
changes: the cell wall had a slightly irregular outline, loosely distributed components and was highly permeable; the cell
wall external layer was more electron dense; the plasmalemma was mostly destroyed and did not adhere to the cell wall;
precipitation of the entire cytoplasm and destruction of organelles and nucleus were seen. Due to these effects, the
morpho-functional relationship between the cell wall and the cytoplasm was destroyed and a less electron dense band
was formed between the altered cytoplasm and the cell wall [33].
The precipitation of the cytoplasm and the destruction of the organelles and nucleus caused the loss of viability and
germination capacity of B. cinerea conidia treated with plant extracts [30]. The antifungal effects of the studied plant
extracts recommend them as good candidates for the in vivo biological control of phytopathogenic fungi, limiting the
overuse of chemical fungicides [40].
6.Conclusions
During the past decades, studies focussing on rose pathogens have greatly increased. Rose diseases caused by the
parasitic fungi P. pannosa, D. rosae, P. mucronatum and B. cinerea can be identified on the basis of the symptoms of
disease and the ultrastructural characteristics of the pathogen. Less is known about the pathogenetic mechanisms.
Because the parasitic fungi limit both productivity and commercial value of roses, disease control strategies demand the
extension of nonchemical disease control and genomic approaches. The principal mechanisms involved include
mycoparasitism, antibiosis, competition and induced resistance. Additional mechanisms include hypovirulence
mediated through fungal viruses, reported for the first time in Botrytis cinerea and enzymatic interference with
pathogenic enzymes [41,42]. Biological control methods seem to be safe and genetic resistance is effective and long
lasting.
Acknowledgements: These studies were financially supported by the Romanian Ministry of Education and Research from the
CNCSIS grants 46/220/2006, 43/220/2007 and PNII–IDEI 2272/2008.
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