Tubakia dryina , symptoms and pathogenicity to Quercus robur

In 1999 disease symptoms on leaves of Quercus robur – necrosis, deformation and blackening of leaf petiole, followed by premature leaf fall were observed. Tubakia dryina was isolated from necrotic tissues and its pathogenicity to oak proved in infection experiments. The fungus caused necrosis of shoots and leaves. Dying leaves displayed also blackening of leaf petiole.


INTRODUCTION
Infection of Quercus robur L. leaves by several fungi results in easily recognizable disease symptoms.Microsphaera alphitoides Griff.et Maubl.causes white powdery mildew on leaf surface (M a ń k a 2005), Taphrina caerulescens (Desm. et Mont.)Tul.
-discoloured spots, raised above and conclave below the blade (E l l i s , E l l i s 1985).Apiognomonia quercina (Kleb.)Hoehn. is causative agent of anthracnose, with blade necroses developed along veins (N e l l y , H i m e l i c k 1967).
There are, however, numerous diseases causing spots on leaves.Their causing agents are difficult to identify due to lack of etiological symptoms, as it is in the case of Septoria quercicola (Desm.)Sacc., Cryptocline cinerescens (Bubak) Arx or Mycosphaerella spp.(G i l m a n , Wa d l e y 1952; B u t i n 1996).Some symptoms, in particular, are not specific as they occur on leaves previously damaged by gall-making insects (B u t i n 1992).
Various symptoms of unknown etiology are common on oak leaves.The conditions favouring their occurrence are not known yet (B u t i n 1996; K o w a l s k i , D u r a k 2000).A few years ago not specific symptoms of this kind were observed on Q. robur leaves, leading to premature defoliation in spring.The symptoms are characterized in the work and results of investigation on the disease etiology are presented.

MATERIALS AND METHODS
Leaves of Q. robur with disease symptoms were collected in June 1999 near Opole (south-west Poland).The symptoms were described in detail and isolations of fungi from necrotic tissues were performed.Leaf disinfection followed the method used for beech leaves by S i e b e r and H u g e n t o b l e r (1987).Leaf bases with petioles were dipped in 96% ethanol (1 min), then in sodium hypochlorite containing ca 4% of active chlorine (3 min) and again in 96% ethanol (0.5 min).After drying in sterile filter paper, from leaf petioles with symptoms 3-mm-long inocula were taken and placed onto 2% malt extract agar (MEA, Difco) in Petri dishes.Incubation for 4 weeks was performed in dark, at room temperature.
The isolation from necrotic tissues yielded almost exclusively Tubakia dryina.Four cultures of the species were grown on MEA and potato dextrose agar (PDA, Difco) at 20 o C, to describe their morphology and to examine and measure their conidiospores (macro-and microconidia).
Infection experiment on leaves was performed on Q. robur trees (ca 10-year-old) in the vicinity of the Ojców National Park of Nature.Isolate No. HMIPC 16 617 was used for inoculation.Developing oak leaves (n = 20) were covered on May 2, 2005, and May 19, 2005, with spore suspension (ca 10 2 viable conidia per ml) obtained from 6-8-week-old cultures grown on MEA.Besides, at the same time, young shoots (n = 6) were wounded with sterile lancet.In the wounds (3-mm-long) going along the shoot an inoculum of ca 5mm 2 of MEA grown with T. dryina mycelium was placed and the shoot covered with parafilm.Control consisted of wounded shoots with sterile medium.Four and eight weeks after inoculation the disease symptoms were evaluated and the fungi isolated from 210 fragments of necrotic leaf tissue, leaf petioles and shoots, as described above.

RESULTS
The disease symptoms on Q. robur leaves observed in 1999 near Opole, were mainly visible on leaf petioles.Necrosis, blackening and deformation by bending down or twisting were extremely characteristic.In some cases the necrosis spread to blade base (Fig. 1).Such leaves fell down prematurely, in spring.From dead leaf petioles almost entirely one fungal species was isolated -Tubakia dryina.Sporadically also fungi representing the genera of Alternaria, Coniothyrium and Epicoccum were isolated.
Over a half of artificially inoculated leaves (65.0%) and all inoculated shoots displayed disease symptoms after 4-8 weeks.Tissue along vein in the upper part of leaves was necrotic (Fig. 5), and some leaves died (Fig. 6).Wounds in the inoculated shoots did not heal, and the tissues around -particularly above wounds, were necrotic and discoloured brown-black (Fig. 7).In the leaves occurring there, leaf petiole was necrotized and black which resulted in dieback of entire leaves (Figs 7,8).The necrotized leaves were not properly developed and displayed atrophy (Fig. 6).In the necrotic tissue T. dryina only sporadically produced conidiomata of pycnothyrium type.

DISCUSSION
Tubakia dryina [syn.Actinopelte dryina (Sacc.)Hoehn.]occurs in North America, Europe and Asia, while its teleomorph, described as Dicarpella dryina Belisario, was  et al. 1996), could have resulted from infection of very young developing leaves of Q. robur.That early artificial inoculation was aimed at finding out if T. dryina could cause necrosis together with malformation and blackening of leaf petioles, leading -according to the 1999 observations -to premature leaf dropping.The results confirmed the hypothesis, which is of interest as the symptoms described were never observed in connection with T. dryina infection.They also pointed to the possible effect of bacteria inhabiting Q. robur leaves on development of T. dryina.
described only in Italy (H o l d e n r i e d e r , K o w a l s k i 1989; P r o f f e r 1990; B el i s a r i o 1991; B u t i n 1996; K a n e k o , K a n e k o 2004).It is found mainly on oaks, and rarely on other plants of Acer, Castanea, Liquidambar, Photinia, Sasafras and Ulmus genera (P r o f f e r 1990).It was described in Poland only once -by P r z y b y ł (1995).In plants the species is able to occur as endophyte, causing no disease symptoms (B o d y , Ra y n e r 1984; P r z y b y ł 1995; C o h e n 1999; G e n n a r o et al. 2003; K a n e k o , K a n e k o 2004).It is also being connected with disease symptoms on leaves and, rather seldom, on shoots (H o l d e n r i e d e r , K o w a l s k i 1989; B u t i n 1996).On Quercus spp.leaves the symptoms are circular, tan to dark reddish brown spots, 1-15 mm in diameter.Small spots may coalesce to form large irregular blotches (H e p t i n g 1971; M u n k v o l d , N e e l y 1990; P r o f f e r 1990).On Castanea leaves brown necrotic spots reach up to 9 mm in diameter and the lesions are surrounded by a prominent chlorotic halo, up to 6 mm wide (E l -G h o l l et al. 1996).Pathogenicity tests with T. dryina yielded inconsistent results.Artificial inoculation of Q. phillyraeoides, Q. glauca and Castanea pubinervis performed by Yo k o y a m a and Tu b a k i (1971) revealed no pathogenicity.The T. dryina isolate examined by H o l d e n r i e de r and K o w a l s k i (1989) did not display any pathogenicity towards shoots and very low pathogenic potential on leaves of Q. robur.On the other hand, E l -G h o l l et al. (1996), K i m and Wa g n e r (1997), and M u n k v o l d and N e l l y (1990) confirmed pathogenicity of the species.The T. dryina isolate tested by the author proved also pathogenic.Despite the fact that it was not morphologically different from the isolates investigated by other authors (Yo k o y a m a , Tu b a k i 1971; H o l d e n r i e d e r , K o w a l s k i 1989; P r o f f e r 1990), it caused different type of symptoms on leaves -a very specific necrosis and blackening of leaf petioles, and necrosis of leaf blade -at first along veins and than of the entire leaf.The necrosis spread so quickly that the leaves did not grow to normal size.The symptoms, very different from those observed by other authors (M u n k v o l d , N e l l y 1990; P r o f f e r 1990; E l -G h o l l