Mycobiota of Castanea sativa Mill. in Azerbaijan - WSL

For. Snow Landsc. Res. 76, 3: 405–408 (2001)
Mycobiota of Castanea sativa Mill. in Azerbaijan
Dilzara N. Aghayeva
Institute of Botany, Azerbaijan National Academy of Science, Patamdart sh. 40, 370073 Baku,
Azerbaijan*
adilzara@hotmail.com
Abstract
During the last few years chestnut (Castanea sativa Mill.) has displayed some signs of degradation
in Azerbaijan. A number of parasitic saprophytic fungi have been noted: Sphaeropsis castaneae,
Diplodia castaneae, Phoma endogena, Cylindrosporium castaneae, Coryneum kunzei, Cladospo -
rium sp., and Mycosphaerella sp.
Ceratocystis castaneae (s.l.) was found frequently in young and old drying chestnuts. In this
paper the anamorph stages of C. castaneae (Sporothrix and Graphium) are described for the first
time. The optimum growth temperature of this fungus is 28–30 °C, the most favourable relative
humidity 90–100%, and the optimum pH 6–6.5. The fungus develops well in media containing
saccharose, maltose or glucose as carbon sources and ammonium phosphate, urea or asparagine
as nitrogen sources.
Keywords: chestnut, Ceratocystis castaneae (sensu lato), Sporothrix, Graphium, dieback
1 Introduction
The fungal pathogens and saprobes of chestnut (Castanea sativa Mill.) have been widely
discussed during the last few years. The fungi Cryphonectria parasitica (Murr.) Barr,
Phytophtora cambivora (Petri) Buism. and Ph. cinnamomi (Rands.) have been recorded as
the most dangerous agents in Europe (ROBIN and HEINIGER this volume; VANNINI and
VETTRAINO this volume). Some fungal species namely, Melanconis modonia (Tul.) Hohn,
Cryphonectria radicalis Fries and Mycosphaerella maculiformis (Pars.) Scherut are also
known to cause damage to chestnut (DIAMANDIS 2001). Cylindrosporium castaneae (Lev.)
Krenner, Cytospora intermedia Sacc., Pseudovalsa madonia (Tul.) Hohn., Coryneum
modonium (Sacc.) Crifon et Maubl, and Phomopsis castaneae Woronich have also been
frequently found on chestnut (JUHÁSOVÁ 1999).
In the second half of the last century the following fungal species have been recorded on
chestnut in Azerbaijan: Microsphaera alphitoides Griff et Maubl, Microsphaerella maculi -
formis (Pars.) Scherut, Cylindrosporium castaneae (Lev.) Krenner, Phyllosticta castanea Ell.
et Ev., Diplodia castanea Sacc., and Leptothyrium castanea Sacc., Cylindrosporium castaneae
was widely distributed and caused considerable damage to the chestnut in the period of 1951
to 1954 (AKHUNDOV 1962).
Pure chestnut forests are rare in Azerbaijan. Fragments of mixed forests occur along the
south macroslopes of the Great Caucasus at 550–1200 m a.s.l. In Minor Caucasus chestnut
spreads over 550 to 600 km with small fragments including pure chestnut plantations and
mixed forests. Chestnut usually grows together with Quercus iberica Stev., Fagus orientalis
Lipsky, Carpinus caucasica Grossh., but rarely with Acer campestre L., and other broad -
leaved species. In recent years chestnut in Azerbaijan has displayed signs of degra dation.
* Present Address: University of Pretoria, FABI, 74 Lunnon Road Hillcrest, ZA-Pretoria 0002, Republic
of South Africa
405

406 Dilzara N. Aghayeva
A drying process has been observed in chestnuts in forests, plantations and private plots in
the Great Caucasus. The intensity of dieback is 45 to 50%. The purpose of this study was to
investigate the mycobiota involved in chestnut dieback.
2 Materials and methods
The fungi were isolated from drying chestnut trees in mixed forests or chestnut plantations that
were 30 years old or older. Leaves, dead and living branches and xylem from trees attacked by
bark beetles were observed under the microscope (MBR-3). Specimens in which it was suspected
that species of Ceratocystis were growing, but not yet sporulating, were placed in a damp chamber.
Fungal isolations were made by transferring spore masses to MEA (20g malt extract, 20g agar per
L water). Identification was made according to POTLAYCHUK and SHEKUNOVA (1985). Mor -
phological traits in culture, optimum pH and temperature optimum were determined according to
BILAY and ELLANSKAYA (1982). The growth of the fungus was studied in liquid glucose-mineral
medium with the pH ranging from 4.5 to 7.0 at 28 °C. The growth was measured by weighing the
dry biomass. Optimum growth temperature was defined on MEA by measuring the diameter of
the colonized area (in mm). The effect of humidity on the germination of spores was studied
according to KHOKHRYAKOV (1976) in the exsiccator with humidities of 50, 60, 70, 80, 90 and
100%. Spores on the dry glass slide were placed in the exsiccator at the optimal temperature of
28 °C. All experiments were performed three times. The carbohydrate and nitrogen requirements
of the fungus were studied on Czapek’s agar (20g sucrose, 2g NaNO 3, 1g KH 2PO 4, 0.5g MgSO 4
7H 2O, 0.5g KCl , 0.01 g FeSO 4 per L water) with a minor modification (BILAY 1978): Sucrose was
replaced by 20g maltose, glucose, cellobiose or cellulose each. 0.4% of NaNO 3, NH 4NO 3,
(NH 4) 3PO 4, urea or asparagin were used as nitrogen sources. Media without any carbohydrate
and nitrogen sources served as controls. The growing temperature was 28 °C.
3 Results
The first symptoms of chestnut drying appeared in June. Leaves became red, gradually
turned yellow, dried out and fell off. Drying began from the top of trees. At first thin, one to
two year old summer branches died back, then the thicker branches. The process of drying
spread down along the crown gradually. Dark brown lines and dark or brownish wood
elements were noted under the bark of the drying trees. Chestnut drying is chronic in nature
and complete drying out of trees can be observed within 4 to 10 years. This drying has been
noted since 1981 in Great Caucasus. The following fungal species were recorded on the
affected chestnut wood, twigs and leaves: Ceratocystis castaneae (Vanin et Solov.) C.
Moreau, Sphaeropsis castaneae Togn., Diplodia castaneae Sacc., Phoma endogena Speg.,
Cylindrosporium castaneae (Lev.) Krenner., Coryneum kunzei Cda., Cladosporium sp., and
Mycosphaerella sp. Cryphonectria parasitca was noted only once in 1942 in Azerbaijan. Since
then it has no longer been found (unpublished data).
Ceratocystis castaneae was found under the bark of drying chestnuts in the Gabala forest
for the first time in 1981 and again in 1994 (GUSEYNOV and AGHAYEVA 1997). Since 1994 it
has been observed in other forests too (mainly in Great Caucasus). Perithecia develope
abundantly on xylem. In culture, colonies are fast growing, covering a petri dish in 15 days at
25 to 30 °C. In the beginning the colonies are white, later becoming white to grey. Both
Sporothrix (Hektoen et Perkins) and Graphium (Cda.) types anamorphs have been recorded.
The conidiophores of Sporothrix anamorphs are clearly branched, hyaline, septate, 33.6–47.6
(42.9) x 2.5–2.8 (2.4) µm. The conidiogenous cells arise terminally on conidiophores as
sympodial denticles. Conidia are holoblastic, hyaline, thin-walled, unicellular, oblong to
ellipsoidal, clavate, 2.8–5.6 x 1.1–1.5 µm. They are formed singly, later becoming aggregated
in a slimy mass. The coremia of the Graphium type are brown, black, pale brown or subhya-

For. Snow Landsc. Res. 76, 3 (2001)
line toward the apex, 240–504 µm long including the head, 36–84 µm in diameter at the base,
and 36–72 µm near the head. The diameter of the head is 120–210 µm. Conidia are onecelled,
hyaline, oblong-cylindrical, sometimes curved, 4.2–8.4 (9.8) x 2.1–2.8 µm accumulating
in slimy heads. In culture coremia formed abundantly in concentric circles. Ascocarps were
produced on the 35 th to 38 th day on MEA after keeping the culture at a low positive
tempera ture (8–12 °C) for the first 10 days. They are numerous, arranged in concentric
circles, innate to superficial, dark brown, 132–180 µm in diameter, and covered by septate,
dark brown hairs, which are 48–75 µm in length and 3–5 µm thick at the base. Necks of the
ascocarps are 1200–1700 µm long, 24–36 µm at the base and 12–14.4 µm thick at the top, with
pale cilia, 24–48 µm long. Asci are evanescent. Ascospores are hyaline, unicellular, commalike,
4.5 x 6.5 µm. They aggregate in a slimy mass at the top of the neck and flow down on it.
This description does not fit the literature completely, and there are several differences to
the description by POTLAYCHUK and SHEKUNOVA (1985) (Table 1).
The development of C. castaneae has been studied under laboratory conditions. Optimum
growth temperature was 28–30 °C. The growth of fungus ceased at temperatures below of
5 °C and above 40 °C. The most favourable humidity for spore germination was 90–100%.
High humidity and high temperatures were not lethal to the fungus, but low humidity at high
temperatures was lethal. Optimum growth was at pH 6–6.5. Tests on the use of carbohydrate
and nitrogen sources showed that the fungus assimilated common sugars easily and de -
veloped well in the media containing sucrose, maltose or glucose. The growth of cultures on
cellulose and cellobiose was near the control without a carbohydrate source. The results also
showed that the fungus assimilated (NH 4) 3PO 4 and organic nitrogen better than NaNO 3.
Table 1. Comparative data for Ceratocystis castaneae (in µm).
Ascocarp Hairs at the base Necks Ostiolar hyphae Ascospores
in POTLAYCHUK
and SHEKUNOVA 84–42 x 68–85 204–347 1000–1800 14.7–20 4.4–7.4
our observations 132–180 48–75 1200–1700 24–48 4.4–6.5
4 Discussion
All the recorded species (Sphaeropsis castaneae Togn., Diplodia castaneae Sacc., Phoma
endogena Speg., Cylindrosporium castaneae (Lev.) Krenner., Coryneum kunzei Cda.,
Cladosporium sp., Mycosphaerella sp.) except C. castaneae (Vanin et Solov.) C. Moreau are
considered to be rare on chestnut. This last species is often found on drying chestnut.
SOLOVYOV (1932) first described the fungus C. castaneae under the name Ceratostomella
castaneae (Vanin et Solov.), as a saprotroph causing a blue stain in the wood of Castanea sp.
Later the fungus was assigned to the genus Ophiostoma and noted as Ophiostoma castaneae
(Vanin et Solov.) Nannf. (cited in POTLAYCHUK and SHEKUNOVA 1985). It has also been
included in the genus Ceratocystis (MOREAU 1952). HUNT (1956) noted that the species may
be imperfectly known because he was unable to obtain material. Then the species was
classified as C. castaneae by POTLAYCHUK and SHEKUNOVA (1985). They classified the genus
Ceratocystis and divided the species of the genus into three groups, with C. castaneae
ascribed to the 3rd group. These authors believe that the species may have either completely
lost its ability to form anamorph sporulation or no sporulation has been dis covered yet.
Their description of the species is very brief. My description partly corresponds to the
literature, but in addition describes anamorph stages (Sporothrix and Graphium) that have
not previously been found. UPADHYAY (1981) does not mention the species. C. casta neae was
considered synonymous with Ceratostomella castaneae (Vanin et Solov.) by SEIFERT et al.
(1993). The systematic state has still to be examined in detail.
407

408 Dilzara N. Aghayeva
According to POTLAYCHUK (1957) the species of the genus Certocystis occurring in oak
may originate from elm. By infecting maple it can be passed back to oak and elm again. In
Azerbaijan the agent of oak decline was established as C. roboris and GUSEINOV (1984)
experimentally proved its pathogenicity. Internal and external signs of drying chestnut trees
correspond to similar symptoms of oak decline. It remains an open question whether the
C. roboris has passed from oak to chestnut and then adapted to it.
It is supposed that C. castaneae takes part in drying of chestnut trees but inoculation tests
should be done to prove the pathogenicity of the fungus.
Acknowledgements
I am grateful to Dr. Ursula Heiniger of the Swiss Federal Institute for Forest, Snow and Land -
scape Research WSL for helpful comments on revising this article.
5 References
AKHUNDOV, T.M., 1962: The biology of the fungus causing silindrosporioz of chestnut. News of
the Azerbaijan Academy of Science, ser. biol. sci. 2: 17–23. (In Russian)
BILAY, V.I., 1978: Culture media. In: The methods of experimental mycologi. “Naukova Dumka”
Kiev, p. 32. (In Russian)
BILAY, V.I.; ELLANSKAYA I.A., 1982: Fundamental mycological methods in phytopathology. In:
Methods of experimental mycology. Kiev. Naukova Dumka. 109: 423–431. (In Russian).
GUSEYNOV, E.S., 1984: Vascular dieback of oak in Azerbaijan J. Micologiya and fitapatologiya L.,
18, 2: 144–149. (In Russian)
GUSEYNOV, E.S.; AGHAYEVA, D.N., 1997: The morphological and cultural signs of the vascular
mycosis agents of forest tree types in Azerbaijan. Tr. J. of Botany 21: 19–26.
DIAMANDIS, S., 2001: The mycoflora of the chestnut coppice ecosystems in Greece. COST G4
Multidisciplinary Chestnut Research. Abstracts of Final Meeting. Switzerland. 47.
KHOKHRYAKOV, M.K., 1976: Methods on experimental study of phytopathogens VIZR.
Leningrad. 65 pp. (In Russian)
JUHÁSOVÁ, G., 1999: Hubove choroby gastana jedleho (Castnea sativa Mill.) VEDA, Bratislava.
191 pp.
HUNT, J., 1956: Taxonomy of the genus Ceratocystis. Lloydia 19: 58 pp.
MOREAU, C., 1952: Coexistence des formes Thielaviopsis et Graphium chez une souche de
Ceratocystis major (van Beyma) nov. conb. Rev.Mycol. (Paris), Suppl. Col. 17: 17–25.
POTLAYCHUK, V.I., 1957: Biology of the agent of oak decline. Proceedings of VIZR. Issue 8,
Stavropol. 227–237. (In Russian)
POTLAYCHUK, V.I.; SHEKUNOVA, Y.G. 1985: Distribution of the species of the genus Ceratocystis
Ell. et Halst. emend Bakshi in USSR. Nov. Sys. Nizsh. Rast. L., Nauka 22: 148–156
ROBIN, C.; HEINIGER, H., 2001: Chestnut blight in Europe: Diversity of Cryphonectria parasitica,
hypovirulence and biocontrol. For. Snow Landsc. Res. 76, 3: 361–367.
SEIFERT, K.A.; WINGFIELD, M.J.; KENDRICK, W.B., 1993: Species of uncertain status. A nomenclature
for described species of Ceratocystis, Ophiostoma, Ceratocystiopsis, Ceratostomella and
Sphaeronaemella. In: Ceratocystis and Ophiostoma. Taxonomy, Ecology and Pathogenicity. St.
Paul Minnesota, APS Press. 269–287.
SOLOVYOV, F.A., 1932: Some rare and little-known species of fungi of North Caucasus region.
Proceedings on Plant Protection. 5, 1, 1. (In Russian)
UPADHYAY, H.P., 1981: A monograph of Ceratocystis and Ceratocystiopsis. Athens, University of
Georgia Press. 176 pp.
VANNINI, A.; VETTRAINO, A.M., 2001: Ink disease in chestnuts: impact on the European chestnut.
For. Snow Landsc. Res. 76, 3: 345–350.
Accepted 4.3.02