Morphological and Molecular Characteristics of the Oak Tree Canker Pathogen, Annulohypoxylon truncatum.

Cankers are localized dead areas in the bark of stems, branches or twigs of many types of trees and shrubs, and are usually caused by fungi. We observed severe canker symptoms in oak trees located in Gyeongnam province in 2011. A total 31 trees were discovered with cankers of varied size, with an average of 48.5 × 15.2 cm. Black, half-rounded globular mound shaped stromata were associated with the cankers, and the asci of the fungi associated with the cankers were cylindrical shaped with their spore-bearing parts being up to 84 µm in length. The average fungal ascospores size was 7.59 × 4.23 µm. The internal transcribed spacer sequence for the canker causing fungus showed 99% similarity to the sequence of Annulohypoxylon truncatum. In this study, the isolated fungus was precisely described and then compared with fungi of similar taxa.

The oak tree colony observed in Sancheong-gun, Gyeongnam province, consisted of a total of 44 oak trees. Of these trees, 15 were Querces variabilis and 29 were Q. acutissima. Thirty-two of the 44 oak trees exhibited severe canker disease symptoms. The sap from the canker affected trees appeared with dark brown discoloration. Fungal stromata were associated with the cankers (Fig. 1A). The stromata bodies were dark brown to blackish brown in color with a glomerate shape (Table 1, Fig. 1A). Most of the stromata developed individually, but some were aggregated. The position of the fungal ostioles was higher than the surface of the stromata. Inside the stromata, a multitude of perithecia were observed. The perithecia were of the spherical type which included a layer of carbonaceous tissue enclosing the perithecia (Fig. 1B). The fungal asci lengths were 96~154 µm in total, with the spore-bearing parts being 85 µm length on average (Table 1, Fig. 1C) [1, 2]. The ascospores were typically hyaline, unicellular, and ellipsoid-inequilateral in shape with no ornamental surface. The size of the ascospores were 7.6 × 4.2 µm on average (Table 1, Fig. 1D). The Q-value of the spores were 1.79, which indicated that they were ellipsoid in form. Based on the fungal morphological characteristics we suspected it to be Annulohypoxylon truncatum.

Fig. 1

A, The stromata as appearing inside a canker; B, Cross section of the stromata; C, Ascus of Annulohypoxylon truncatum; D, The ascospores were hyaline and one-celled (scale bars: C = 50 µm, D = 10 µm).

Table 1

Comparison of the morphological characteristics of A. truncatum observed in this study

The fungi Annulohypoxylon spp. belongs to the genus, Hypoxylon and family, Xylariaceae. Form-genus Nodulisporium Preuss is an anamorph of Hypoxylon. The Hypoxylon genus has two distinguishable sections, Hypoxylon and Annulate [2]. Those two sections are differentiated by three major characteristics: 1) the presence or absence of a carbonaceous layer in the stromatal tissue, 2) the height of the ostioles (those of Hypoxylon are usually lower than the stromatal surface level and cannot be easily observed, and there is no an annulate disc), and 3) most Hypoxylon species do not have perispores on their ascospores. Hypoxylon spp. have well developed stromata with multiple perithecia and the ascus apical ring stains blue color when an iodine solution is applied. Hypoxylon spores are usually hyaline and single celled. Based on the results of β-tubulin and α-actin gene sequences analyses, Hsieh et al. [3], relocated Hypoxylon into a new genus for which Annulohypoxylon received a new name. The fungi of genus Annulohypoxylon are ubiquitous with numerous reports of their presence in tropical regions, and they identified as oak tree pathogens.

To support the morphological identification of the fungus using molecular analysis tools, we utilized the primers internal transcribed spacer (ITS)1 and ITS4 [4] to amplify and sequence an ITS rDNA region of the isolate. The sequence was deposited in GenBank (accession No. JQ303335). The ITS sequences shared 99% similarity with sequences of the Annulohypoxylon truncatum strain xsd08029 (accession No. FJ478107). Phylogenetic analysis was conducted using MEGA5 with a neighbor-joining algorithm. The ITS sequences of closely related fungi sections, Annulate and Xylariaceae, served as references. Phytophtora ramorum, the pathogen responsible for Sudden Oak Death, was identified as an outgroup taxon (Fig. 2).

Fig. 2

A phylogenic tree generated from the internal transcribed spacer sequences using the MEGA5 program with the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches (1,000 replicates). All taxa in the tree belonged to the Family Xylariaceae, except Phytophthora ramorum, which is a Sudden Oak Death pathogen. P. ramorum was used as an outgroup taxon. The present isolate was marked with a black diamond, ♦.

Lee and Whalley [5, 6] fully described the genus Hypoxylon as it occurred in Asia. They reported observing H. acrhri, H. bovei, H. stygium, H. fragiforme, H. fuscum, H. howeianum, H rubiginosum, H. subgilvum, H. crocopeplum, and H. jecorinum, in Asia. However, A. truncatum had not been clearly described in Korea. In this study, we described the characteristics of the A. truncatum OC isolate in terms of its morphological and molecular aspects.