Draženka Jurković

First Report of Diaporthe phaseolorum on Sunflower (Helianthus annuus) in Croatia

Sunflower (Helianthus annuus L.) is a crop that is grown worldwide for the production of edible oil. In Croatia, it has considerable economic significance. From 2004 to 2007, sunflower stems showed light-to-dark brown lesions of different sizes and shapes. The lesions were observed for the presence of pycnidia in affected areas. Isolations from infected tissue on potato dextrose agar (PDA) yielded in two fungal species. One, which was isolated in most cases, was the well known sunflower pathogen Diaporthe helianthi Munt. Cvet. Morphological characteristics, stromata pattern, formation of alpha and beta conidia, and ascostromata characteristic of the other isolated fungus matched the description of D. phaseolorum (Cooke & Ellis) Sacc. (2). D. phaseolorum frequency was 5%. On PDA, the fungus formed white, floccose, aerial mycelium that filled a petri dish (9 cm) in 6 days. D. phaseolorum produces conidiomata in black stromatic structures, which consist of pycnidia with alpha and beta conidia. The alpha conidia were unicellular, hyaline, ellipsoidal to fusiform, and 5.6 to 10.0 × 1.9 to 4.8 μm. The beta conidia were hyaline, elongated, filiform, straight, curved at one or both ends, and 11.7 to 27.6 × 0.7 to 2.0 μm. After 50 days, perithecia were formed. Asci were clavate and 27.64 to 40.1 × 5.70 to 8.2 μm. Eight ascospores formed within asci. Ascospores were two-celled, elliptic, hyaline, and slightly constricted at the septa, and 8.93 to 13.5 × 2.1 to 4.0 μm. Amplification and sequencing of the internal transcribed spacer (ITS) rDNA region were performed with ITS4 and ITS5 universal primers (3) on two isolates (Su9 and Su10) and data were deposited in GenBank (Accession Nos. GQ149763 and GQ149764). Comparison of sequences available in GenBank revealed that the ITS sequence was identical to D. phaseolorum found on Stokesia laevis Hill (Greene) (U11323/U11373) and identical to the strain CBS 116020 isolated from Aster exilis Elliot. (AY745018). On the basis of the obtained results of morphological characteristics and molecular approaches, the pathogen was identified as D. phaseolorum. Pathogenicity evaluation consisted of artificial infections on field-grown sunflower plants at the full button stage as described by Bertrand and Tourvielle (1). A leaf test was done by placing a mycelial plug of 5 × 5 mm from a cork borer of two isolates (Su9 and Su10) on the tip of the main vein. The inoculation site was covered with moistened, cotton wool and wrapped in aluminum foil to prevent the inoculum from drying out. Ten plants of each of the four replications were inoculated. Control plants were inoculated with pure PDA plugs. Lesions of 12 to 40 mm long were observed on the sunflower leaf 10 days after inoculation. Control plants did not develop symptoms. The pathogen was reisolated from the infected plants. To our knowledge, this is the first report of the finding of D. phaseolorum on sunflower in Croatia and we have no literature data about the occurrence of this fungus on sunflower in the world. References: (1) F. Bertrand and D. Tourvielle. Inf. Tech. CETIOM 98:12,1972. (2) E. Punithalingma and P. Holliday. No. 336 in: Descriptions of Pathogenic Fungi and Bacteria. CMI/CAB, Kew, Surrey, England, 1972. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

First Report of Cane Blight on Blackberry Caused by Diaporthe eres in Croatia

A cane disease of blackberry (Rubus sp.) cv. Thornfree was observed in May and June 2010 in two growing regions in the eastern part of Slavonia in Croatia. Symptoms consisted of bleached areas between and around cane nodes with some canes showing wilt symptoms. Infected areas were covered with numerous, black pycnidia immersed in the epidermal tissue. Disease occurrence in orchards growing cv. Thornfree ranged between 1 and 15%. Thirty disease samples were collected, disinfected (1 min in 70% ethanol and 2 min in 1% NaOCl), and placed in a moist chamber for 4 days. Fungal sporulating structures were then picked off and placed on potato dextrose agar (PDA). Fungal isolates obtained were identified as a Phomopsis sp., the conidial state of Diaporthe (3), on the basis of cultural and morphological characteristics. Alpha conidia were unicellular, hyaline, fusiform, sometimes tapering toward one or both ends, biguttulate (sometimes with several guttules), and 5.2 to 9.7 × 1.4 to 2.7 μm (average 6.5 × 2.1 μm). Beta conidia were hyaline, aseptate, filiform, hamate, and 16.6 to 28.2 × 0.5 to 1.5 μm (average 24.0 × 1.1 μm). The teleomorph was not observed. Biomolecular analysis was performed to identify the fungal species by sequencing the internal transcribed spacer (ITS) region spanning ITS 1, 5.8S rDNA, and ITS 2 of two isolates (Phk1 and Phk2). The amplified product was sequenced (GenLab-Enea, Rome, Italy) and a BLAST search of the NCBI nucleotide database was performed. Sequences from Phk1 and Phk2 (GenBank Accession Nos. HQ533144 and HQ533143, respectively) were identical to authentic and vouchered Diaporthe eres Nitschke (GenBank DQ491514, BPI 748435, and CBS 109767) ITS sequences in GenBank. Fungal isolates for pathogenicity tests were grown on PDA at 25°C for 7 days (12 h light/dark regimen). Inoculations were made on 30 to 40 cm long green shoots of potted plants of the blackberry cv. Thornfree. One-centimeter long wounds were made with a sterile scalpel and mycelia of D. eres were placed in the wounds. Inoculation sites were covered with a piece of wet cotton wool and aluminum foil to retain moisture. Three replications of 10 plants each were inoculated and these plus 10 control plants (inoculated with plugs of PDA only) were maintained in a growth chamber at 25°C. After 25 days, lesions developed on all 30 inoculated plants that averaged 15 mm long and control plants remained symptomless. D. eres was reisolated from inoculated plants, thus completing Kochs postulates. Phomopsis spp. have previously been reported on blackberry canes in Serbia (1) and Yugoslavia (2,4), however, to our knowledge, this is the first report of the occurrence of D. eres (anamorph P. oblonga) on blackberry in Croatia. References: (1) M. Arsenijevic. Biljni Lekar 34:117, 2006. (2) M. Muntanola-Cvetkovic et al. Zast. Bilja 36:325, 1985. (3) B. C. Sutton. Page 569 in: The Coelomycetes. CMI, Kew, Surrey, UK, 1980. (4) M. Veselic et al. Zast. Bilja 49:76, 1998.

First report of Phomopsis longicolla on Cocklebur (Xanthium strumarium) in Croatia

Cocklebur (Xanthium strumarium L.; family Asteraceae) is a widespread weed species in eastern Croatia found especially in arable crops including soybean. Symptoms of disease appear after the plants reach physiological maturity. Stems and branches are completely blighted, and on the surface, are covered with minute, black pycnidia embedded in the epidermal tissue of the host and are especially numerous around nodes. More than 100 plants with symptoms were examined. From each plant with symptoms, three pieces of symptomatic tissues (5 to 10 mm) were disinfected and placed on potato dextrose agar (PDA), pH 4.5 and 25°C with a 12/12 h of light/dark regimen. The cultural and morphological characteristics of the fungi isolated from X. strumarium corresponded with those described (1) for Phomopsis longicolla Hobbs isolated from soybean. P. longicolla frequency was 3%, while other isolates belonged to other Phomopsis species. To confirm the morphological identification of isolates, molecular identification was performed. DNA of four isolates was extracted from 7-day-old monoconidial cultures grown on PDA. The internal transcribed spacer (ITS) regions of ribosomal DNA were amplified with universal primer ITS4 and ITS5 and sequenced (M-Medical Genenco, Rome). The sequences were aligned with the multiple sequence alignment program ClustalW, showing 100% similarity among them, and a sequence (GenBank Accession No. EF026104) was compared with the ITS sequences available on the database, revealing that it is identical to many P. longicolla isolates. To confirm Kochs postulate, cocklebur plants were infected in the field by applying mycelial plugs (5 mm in diameter) from the margin of 6-day-old cultures to the plant stem. The inoculation point was internodal at the mid-stem. After inoculation, plugs were covered with a piece of cotton wool and aluminum foil. Stem lesions were measured 10 days after inoculation. Mean stem lesions were 15 to 21 mm. A pathogenicity test was also done on soybean cv. Tisa (21-day-old) seedlings by applying mycelium plugs (5 mm) with a sterile scalpel on previously wounded hypocotyls. The inoculate point was covered with wet cotton wool and aluminum foil. After 10 days, mean stem lesions were 18 to 30 mm. The pathogen was always reisolated from the stem lesions. Control plants inoculated by PDA plugs did not exhibit any symptoms. There is a report of P. longicolla on cocklebur in the United States (2) and on other plants from the Asteraceae family. Other weeds such as Abutilon theophrasti Med., were shown to be a host of fungal pathogens belonging to the Phomopsis/Diaporthe complex of soybean (3). Our results also confirm that cocklebur could be a natural inoculum source for Phomopsis seed decay of soybean caused primarily by P. longicolla. However, to our knowledge, this is the first report of P. longicolla being isolated from naturally infected cocklebur in Croatia. References: (1) T. W. Hobbs et al. Mycologia 77:535, 1985. (2) K. W. Roy et al. Can. J. Plant Pathol. 19:193, 1997. (3) K. Vrandecic et al. Plant Pathol. 53:251, 2004.

Influence of Substrate in Biological Control of Tomato Seedlings against Rhizoctonia Solani and Pythium Debaryanum

One of the biggest challenges for the safe maize production is the yield stability in a wide range of environments with different soil fertility, weather conditions, prevailing pests and diseases as well as cultural practices. In order to achieve it, new hybrids with higher tolerance to biotic and abiotic stress are continuously being created. In order to compare performance of maize hybrids in environments with different levels and types of stress, and relate it to the stalk lodging incidence, we compared performance of 64 maize hybrids obtained by crossing 16 inbred lines with four inbred testers in three different environments. Two environments at same location (Rugvica) differed in crop rotation, and the third environment at location Botinec was considered as dry because of low water capacity of the soil. Mixed models that included all design elements and genetic background of hybrids were used to analyze the effect of lodging on yield in different environments. The results show that stalk lodging had a significant effect on yield, but significant lodging x environment interaction indicates that this effect was rather environment specific.Sweet pepper Istra F1 hybrid was grown in a greenhouse on K rich soil. For evaluation of K antagonism, two K rates (55 and 85 kg ha-1) were applied in a randomized complete block design with four replications. Total number of fruits, total number of BER affected fruits as well as average fruit mass were recorded for each harvest. Fruit and leaf K and Ca content were determined at three plant growth stages (at the stage of the first, third and fifth fruit cluster). Higher K rate gave higher : average fruit mass (169.45 g), total number of fruits per plant (7.95), number of BER affected fruits per plant (3.82), K in leaves (5.44% DM), K in fruits (6.35% DM), but negatively correlated with Ca concentration in fruits (0.57% DM) and leaves (3.24% DM). This resulted in decreased marketable yield of sweet pepper fruits.