Diplodia species are known as pathogens on many woody hosts, including fruit trees, worldwide. In this study a collection of Diplodia isolates obtained mostly from apple and other Rosaceae hosts were identified based on morphological characters and DNA sequence data from ITS and EF1-α loci. The results show that the diversity of species associated with twig and branch cankers and fruit rot of apples is larger than previously recognised. Four species were identified, namely D. seriata and D. malorum (which is here reinstated for isolates with D. mutila-like conidia). Diplodia intermedia sp. nov. is closely related to D. seriata, and D. bulgarica sp. nov. is morphologically and phylogenetically distinct from all Diplodia species reported from apples.

In the late summer of 2005, sporadic and unusual damage was observed on pepper (Capsicum annuum cv. Kurtovska kapia and local cv. Ribka) on two farms and tomato (Lycopersicon esculentum cv. Florida 47) fruits on one farm in the Plovdiv Region of Bulgaria. Dry, round, sunken zones (10 to 20 mm) were observed on pepper fruits that preserved their natural skin color even after black acervuli containing orange masses of conidia appeared. Eventually, the lesions turned brown, coalesced, and the fruits mummified on the plants. Tomato fruits developed similar symptoms, with less prominent discoloration and fewer acervuli. The pathogen was easily isolated from both hosts on potato dextrose agar where it formed white-to-gray colonies with salmon orange pigmentation on the reverse side of the plates. Conidia that formed were hyaline, fusiform, aseptate, and measured 13.3 to 17.4 × 3.5 to 5.5 μm and 11.6 to 15.5 × 4.1 to 5.0 μm for pepper and tomato isolates, respectively. Both cultural and morphological characteristics of the isolates were similar to those described for Colletotrichum acutatum (3). Koch's postulates were performed with two representative isolates from each host by artificial inoculation of healthy, green pepper and ripe tomato fruits from the respective cultivars. Fruits were wound inoculated with a sterile scalpel, and small agar plugs (3 to 4 mm) containing 7-day-old sporulating cultures were placed on each wound (five fruits per isolate), or by pipette tip-pricking and pipetting a 5-μl droplet of a conidial suspension (5 × 106 conidia ml–1) on each wound. The same number of wounded, noninoculated fruits was used as a control. Fruits were maintained in a humidity chamber at 22 to 25°C, and 4 days later, sunken necrotic zones were observed around the wounds of inoculated fruit, whereas control fruits remained symptomless. The pathogen was subsequently reisolated from the inoculated diseased tissues but not from the control fruits. Species-specific PCR (using primer pair CaInt2/ITS4) (2,4) of genomic DNA from three representative isolates (two from pepper and one from tomato) resulted in an amplification product of 490 bp, specific for C. acutatum, further confirming the identity of the pathogen. To our knowledge, this is the second report of C. acutatum in Bulgaria (1), and the first occurrence of that agent on tomato and pepper in this country. References: (1) S. G. Bobev et al. Plant Dis. 86:1178, 2002. (2) S. Freeman et al. Phytopathology 91:586, 2001. (3) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (4) M. L. Lewis Ivey et al. Plant Dis. 88:1198, 2004.

In the winter of 2007, severe damage was observed on numerous indoor potted plants of Dracaena sanderiana hort. Sander ex Mast. (“lucky bamboo”) in Plovdiv, Bulgaria, which were imported from a country of unknown origin. These plants were already in the retail distribution stream. Initially, the internodes of infected stems appeared pale green with yellowish lesions. An upward spreading necrosis led to a weakness of the stems with wilt and death of the plants occurring within 2 weeks. Eventually, entire stems were covered with numerous, black, globose-to-ellipsoid acervuli with sparse, black setae. The fungus was aseptically isolated from stem lesions on potato dextrose agar (PDA) on which it produced fast-growing, pale pink colonies. On the plant and in culture, the conidia were hyaline, broadly clavate to cylindrical, occasionally slightly curved, and measured 20 to 34 × 6.7 to 10.0 μm (average 28 × 8.5 μm). On the basis of the symptoms on the plant and morphological, cultural, and molecular characteristics, the fungus was identified as Colletotrichum dracaenophilum D.F. Farr & M.E. Palm (1). Pathogenicity of the fungus was confirmed by artificial inoculation of healthy plants of D. sanderiana (three replicates). Stems were inoculated by inserting small mycelial plugs from 7-day-old PDA cultures into wounds that were subsequently covered with Parafilm strips. After 2 weeks, pale green lesions started developing on all inoculated plants and the fungus was successfully reisolated. No symptoms were found around the pure agar control wounds. The specimen from Bulgaria was deposited in the U.S. National Fungus Collections (BPI 877337) with the derived culture deposited as CBS 121453. In addition, the internal transcribed spacer region of the nrDNA of this isolate was sequenced and deposited as GenBank Accession No. EU003533. To our knowledge, this is the first report of C. dracaenophilum on potted plants outside of China and is the first report of this species in Bulgaria. Reference: (1) D. F. Farr et al. Mycol. Res. 110:1395, 2006.

Strawberry anthracnose was first reported in Bulgaria in 2002, subsequently reaching disease epidemic proportions in production fields from 2004. Isolates of Colletotrichum species from different strawberry cultivars (112), pepper (2), tomato (1), and the weed species Cirsium arvense (1), Abuthilon theophrasti (1) and Rumex obtusifolius (1) growing adjacent to cultivated strawberry fields, were characterized by morphological criteria and by various molecular methods. Morphological characterization defined all the isolates as Colletotrichum acutatum, which was ultimately confirmed by species‐specific polymerase chain reaction (PCR) analysis. No PCR products were amplified using Colletotrichum gloeosporioides specific primers. Arbitrarily primed PCR determined that all the studied isolates, regardless of which plant species they originated from, were uniform indicating that they belong to an asexually reproducing population. Sequence analysis of the complete ITS (ITS 1 – 5.8S – ITS 2) region further identified the pathogen populations as C. acutatum and grouped them within the previously defined subgroup II, ‘Ca‐clonal’, which is uniform. Pathogenicity tests and alternate host infections imply that cross‐inoculation of C. acutatum between strawberry and other susceptible hosts is occurring under field conditions.

Recently, there has been increasing interest in growing strawberries (Fragaria × ananassa) in Bulgaria. During the spring seasons of 2001 and 2002, progressive fruit damage in some producers' plots was observed in the Plovdiv Region. Appearance and development of symptoms were typical of anthracnose on aboveground plant structures. On immature fruits, lesions were single or in groups of two or three, circular, small (1 to 3 mm), sunken, and dark brown to black. Lesions on ripe fruits were similar in shape and color, larger (10 to 15 mm), and when coalesced, they covered most or the entire surface. Elliptical, sunken lesions with lighter centers were also found on petioles and stolons, and blighted flowers and infected immature fruits became dark in color. Under wet conditions, salmon-colored masses of conidia were produced mainly on mature but also on unripe fruits, and on petioles and stolons. Conidia were hyaline, cylindrical, and attenuated at both ends measuring 11.5 (9.2 to 13.3) × 4.5 (4.2 to 5.0) μm (2). Colonies of the isolated fungus growing on potato dextrose agar were white with mycelium becoming gray and aerial. Symptoms were reproduced by artificial inoculation of healthy, ripe strawberry fruits. Fruits were wounded with a sterile scalpel, and 5-mm plugs of agar cultures of two isolates were placed on each wound (12 fruits per isolate). An equal number of wounded and noninoculated fruits were used as a control. Fruits were kept in a humidity chamber at 25°C, and 3 to 5 days later, necrotic sunken zones containing acervuli were observed around the wounds of inoculated fruits, and the pathogen was subsequently reisolated. Identical symptoms were obtained after inoculation of raspberry fruits. Intact strawberry stolons and stalks were also successfully inoculated in the lab with two additional isolates using the same technique and tightly covering wounds with adhesive tape. No symptoms were found in the controls of all tests. On the basis of symptoms and pathogen characteristics, the disease was confirmed as anthracnose (black spot) caused by Colletotrichum acutatum. Additionally, the identity of six isolates was confirmed as C. acutatum by species-specific polymerase chain reaction amplification resulting in a single fragment of 490 bp using primers ITS4 and CaInt2 (1). To our knowledge this is the first report of C. acutatum on strawberry in Bulgaria. References: (1) S. Freeman et al. Pathogenic and non-pathogenic lifestyles in Colletotrichum acutatum from strawberry and other plants. Phytopathology 91:986, 2001. (2) P. S. Gunnell, and W. D. Gubler. Taxonomy and morphology of Colletotrichum species pathogenic to strawberry. Mycologia 84:157, 1992.