405 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., andMycosphaerella sp.
Ceratocystis castaneae (s.l.)was found frequently in young and old drying chestnuts. In this paper the anamorph stages of C. castaneae(Sporothrixand 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 HEINIGERthis volume; VANNINIand VETTRAINOthis 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 (DIAMANDIS2001). 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 (JUHÁSOVÁ1999).
In the second half of the last century the following fungal species have been recorded on chestnut in Azerbaijan: Microsphaera alphitoidesGriff et Maubl, Microsphaerella maculi - formis (Pars.) Scherut, Cylindrosporium castaneae(Lev.) Krenner,Phyllosticta castaneaEll.
et Ev.,Diplodia castanea Sacc.,andLeptothyrium castanea Sacc., Cylindrosporium castaneae was widely distributed and caused considerable damage to the chestnut in the period of 1951 to 1954 (AKHUNDOV1962).
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 ibericaStev., Fagus orientalis Lipsky, Carpinus caucasicaGrossh., 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, Repu- blic of South Africa
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 Ceratocystiswere 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 BILAYand 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 NaNO3, 1g KH2PO4, 0.5g MgSO4 7H2O, 0.5g KCl , 0.01 g FeSO4per L water) with a minor modification (BILAY1978): Sucrose was replaced by 20g maltose, glucose, cellobiose or cellulose each. 0.4% of NaNO3, NH4NO3, (NH4)3PO4, 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 parasitcawas noted only once in 1942 in Azerbaijan. Since then it has no longer been found (unpublished data).
Ceratocystis castaneaewas found under the bark of drying chestnuts in the Gabala forest for the first time in 1981 and again in 1994 (GUSEYNOVand AGHAYEVA1997). 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 Sporothrixanamorphs 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 Graphiumtype are brown, black, pale brown or subhya-
407 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 one- celled, 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 35th to 38th 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, comma- like, 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 POTLAYCHUKand SHEKUNOVA(1985) (Table 1).
The development of C. castaneaehas 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 (NH4)3PO4 and organic nitrogen better than NaNO3.
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 castaneaeTogn., 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. castaneaeunder 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 Ophiostomaand noted as Ophiostoma castaneae (Vanin et Solov.) Nannf. (cited in POTLAYCHUK and SHEKUNOVA1985). It has also been included in the genus Ceratocystis (MOREAU1952). HUNT(1956) noted that the species may be imperfectly known because he was unable to obtain material. Then the species was classified as C. castaneaeby POTLAYCHUKand SHEKUNOVA(1985). They classified the genus Ceratocystis and divided the species of the genus into three groups, with C. castaneae ascribed to the 3rdgroup. 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 andGraphium) that have not previously been found. UPADHYAY(1981) does not mention the species. C. casta neaewas considered synonymous with Ceratostomella castaneae(Vanin et Solov.) by SEIFERTet al.
(1993). The systematic state has still to be examined in detail.
408 Dilzara N. Aghayeva
According to POTLAYCHUK(1957) the species of the genus Certocystisoccurring 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. roborisand 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. roborishas passed from oak to chestnut and then adapted to it.
It is supposed that C. castaneaetakes 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.
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Accepted 4.3.02
