Jasenka Ćosić

Resolving the Diaporthe species occurring on soybean in Croatia

Diaporthe (anamorph = Phomopsis) species are plant pathogens and endophytes on a wide range of hosts including economically important crops. At least four Diaporthe taxa occur on soybean and they are responsible for serious diseases and significant yield losses. Although several studies have extensively described the culture and morphological characters of these pathogens, their taxonomy has not been fully resolved. Diaporthe and Phomopsis isolates were obtained from soybean and other plant hosts throughout Croatia. Phylogenetic relationships were determined through analyses of partial translation elongation factor 1-alpha (EF1-α) gene and ITS nrDNA sequence data. By combining morphological and molecular data, four species could be distinguished on soybeans in Croatia. Diaporthe phaseolorum is described in this study and its synonyms are discussed. Diaporthe phaseolorum var. caulivora is raised to species status and the name Diaporthe caulivora is introduced to accommodate it. A species previously known as Phomopsis sp. 9 from earlier studies on sunflower, grapevine, rooibos and hydrangea is reported for the first time on soybean, and is formally described as Diaporthe novem. The well-known soybean pathogen Phomopsis longicolla was also collected in the present study and was transferred to Diaporthe longicolla comb. nov. The presence of these species on herbaceous hosts raises once more the relevance of weeds as reservoirs for pathogens of economically important plants.

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.

Influence of sowing date on the occurrence of fusarium head blight on wheat — A phytosanitary food safety problem

Fusarium head blight is a worldwide disease of small grain cereals, including wheat, which has a negative impact on grain yields and quality. Six winter wheat cultivars were sowing during three years (2000-2002) on 25th September, 15th October and 1st November at the location of Osijek. The grain infection with Fusarium species was determined after harvesting in laboratory using deep-frezing method. The least infection intensity was observed on all cultivars sowed at the end of September, although some cultivars (Zitarka, Srpanjka, Demetra) sowed at mid October did not achived significant differences in grain infection in comparison to those sowed at the end of September. Respecting all three research years and sowing dates, the cv. Zitarka had the smallest number of infected grains

The spreading of Alfalfa mosaic virus in lavandin in Croatia

SUMMARY A survey was conducted in 2012 and 2013 to detect the presence and distribution of Alfalfa mosaic virus (AMV) in lavandin crops growing in continental parts of Croatia. A total of 73 lavandin samples from six crops in different localities were collected and analyzed for the presence of AMV and Cucumber mosaic virus (CMV) using commercial double-antibody sandwich (DAS)-ELISA kits. AMV was detected serologically in 62 samples collected at three different localities, and none of the samples tested positive for CMV. For further analyses, six selected samples of naturally infected lavandin plants originating from different localities were mechanically transmitted to test plants: Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana and Ocimum basilicum, confirming the infectious nature of the disease. Molecular detection was performed by amplification of a 751 bp fragment in all tested samples, using the specific primers CP AMV1/CP AMV2 that amplify the part of the coat protein (CP) gene and 3’-UTR. The RT-PCR products derived from the isolates 371-13 and 373-13 were sequenced (KJ504107 and KJ504108, respectively) and compared with the AMV sequences available in GenBank. CP sequence analysis, conducted using the MEGA5 software, revealed that the isolate 371-13 had the highest nucleotide identity of 99.5% (100% amino acid identity) with an isolate from Argentina originating from Medicago sativa (KC881010), while the sequence of isolate 373-13 had the highest identity with an Italian AMV isolate from Lavandula stoechas (FN667967) of 98.6% (99% amino acid identity). Phylogenetic analysis revealed the clustering of selected isolates into four molecular groups and the lavandin AMV isolates from Croatia grouped into two distinct groups, implying a significant variability within the AMV lavandin population.

First report of foliar and stem blight on sunflower caused by Alternaria helianthiinficiens in Croatia.

Sunflower (Helianthus annus L.) is the most important oilseed crop in Croatia. In August 2009, in six localities of eastern Croatia, severe foliar and stem blight symptoms were observed on several genotypes with disease incidence ranging from 10 to 50%. At the initial stage of the infection, irregular to oval, brown spots different in size, surrounded by a chlorotic halo, appeared on the leaves that gradually became enlarged and coalesced, and whole leaves turned yellow and necrotic, followed by defoliation. Lesions on the stems were light to dark brown, randomly distributed, rounded and tapered on the ends; later becoming large and elongated causing stem breakage. Tissue within the lesion was reddish on the cross section. To determine the causal agent, small pieces of symptomatic leaves and stem tissue of sunflower were surface disinfested and placed on potato dextrose agar. A total of 17 isolates from leaves as well as six from stems were obtained and all formed cottony, dark olivaceous to black colonies under 12 h of fluorescent light per day. All isolates formed uniform solitary, pale brown to brown, long ovoid conidia with five to eight transverse and one to two longitudinal septa. The conidia of all isolates were slightly constricted at the transverse septa, measuring 55 to 90 × 14 to 20 μm. Based on the morphological characteristics, the pathogen was identified as Alternaria helianthiinficiens E.G. Simmons, Walcz & R.G. Roberts (4). The pathogenicity was tested with one representative isolate (Alt5) by injection of a conidial suspension (106 conidia/ml) into stems of 20 healthy sunflower seedlings and by spraying 20 non-wounded detached leaves with a suspension of spores. Small necrotic spots on all inoculated seedlings and leaves formed 5 and 9 days after inoculation, respectively. The control sunflower seedlings and detached leaves, inoculated with sterile water, showed no reactions. The identity of isolate Alt5 was futher confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA. Because there are no available corresponding ITS sequences of A. helianthiinficiens in the GenBank, reference type strain CBS 208.86 (publicly purchased, CBS, Utrecht, Netherlands) was also sequenced in this study. Total DNA was extracted directly from fungal mycelium and PCR amplification and sequencing were performed with primers ITS1F/ITS4. Sequence analysis of ITS region revealed 100% nucleotide identity between isolate Alt5 (GenBank Accession No. JX101648) and isolate CBS 208.86 (GenBank Accession No. JX101649). The nucleotide identity of both isolates compared with A. helianthi (HM449991), another sunflower pathogenic fungus, was only 80%. A. helianthiinficiens has previously been reported on sunflower in Hungary and the USA (3), Serbia (1), and Korea (2). However, to our knowledge, this is the first report of A. helianthiinficiens occurrence in Croatia as a new and harmful parasite of sunflower, illustrating an expansion of its geographical range and underscoring the need for phytosanitary control because it is a seedborne fungus. References: (3) M. Aćimović and N. Lačok. Helia 14:129, 1991. (4) H. S. Cho and S. H. Yu. Plant Pathol. J. 16:331, 2000. (2) E. G. Simmons. Mycotaxon 25:203, 1986. (1) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands, 2007.

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.

Fusarium species isolated from plant debris in eastern Croatia

Maize (Zea mays L.) genotypes wit It improved nitrogen utilization efficiency (NUE) are of interest to growers. Field experiments were conducted to evaluate the total N uptake in the aboveground biomass and NUE (kg grain per kg of N absorbed in the aboveground biomass). Four commercial hybrids of similar maturity ranking were grown under the high-N (200 kg N ha(-1)) and low-N (1100 kg N ha(-1)) fertilization rates over three years. Growing conditions significantly affected hybrid performance for NUE, which ranged from 42 kg grain kg N-1 in the low-yielding (dry) environment to 55 kg grain kg N-1 under higher yielding environment, When compared to the low-N rate, the average N uptake was by 32% higher at the high-N rate, whereas smaller differences occurred for the aboveground biomass (12%), grain yields (14%), stover N (28%) and grain N (13%) concentrations. Significant differences existed among tested hybrids for grain yield, aboveground biomass, grain and stover N concentration, N uptake and consequently NUE.Hairy vetch (Vicia villosa Roth) and crimson clover (Trifolium incarnatum L.) were evaluated as cover crops during two vegetation seasons in the Mediterranean and Continental area of Croatia. Better tolerance to low winter temperatures was observed for crimson clover. Higher plants (10 to 3 1 cm) before winter and in spring (67 to 117 cm) were measured for hairy vetch, while crimson clover had higher yields of fresh biomass (33.7 to 113.1 t ha(-1)). Concentrations of N (1.53 to 3.34%), P2O5 (0.55 to 1.04%) and K2O (2.02 to 5.32%) in plant tissue were higher for hairy vetch. However, due to higher yields of dry matter, crimson clover accumulated more N (105 to 239 kg ha(-1)), P2O5 (28 to 83 kg ha(-1)), and K2O (105 to 440 kg ha(-1)) than hairy vetch. According to our results crimson clover could be recommended as a cover crop in Mediterranean and Continental area of Croatia.

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.

A survey of Fusarium graminearum and deoxynivalenol contamination of malt barley from the crop year 2004 in eastern Croatia

Fusarium graminearum and deoxynivalenol contamination of malt barley were investigated at several locations in eastern Croatia. The mean number of infected grains ranged from 1.05 - 36.13% and the mean mycotoxin content for the three locations were 0.07, 0.08 and 2.19 ppm, respectively.

First Report of Wheat spindle streak mosaic virus on Wheat in Croatia

Wheat spindle streak mosaic (WSSM ; genus Bymovirus, family Potyviridae), transmitted by plasmodiophorid Polymyxa graminis Led., is one of the most important wheat viruses that cause significant yield losses (Deb and Anderson 2008). In March 2013, irregularly distributed, light green to yellow, circular patches in a wheat crop characteristic of soilborne viruses were observed in one local winter wheat (Triticum aestivum L.) cultivar Super Žitarka in the Karanac locality (Baranja Country, Croatia). Initial symptoms included light green to yellow dashes, mottling, and chlorotic, spindle-shaped streaks with green center on the leaves, which became yellow and eventually necrotic. Affected plants were stunted, with dark brown, slightly swollen, and enlarged roots. The long period of wet and cool spring weather most likely favored disease development and severe symptoms were visible by the end of vegetation with estimated incidence of 40%. The roots of infected plants were stained in lactophenol cotton blue and cystosori of P. graminis were observed in all assayed wheat roots by light microscope. Because of that, soil samples were collected from this locality and used in bait plant test. Wheat bait plants showed chlorosis and mild mosaic 6 weeks after sowing, and cystosori were detected in their roots. Using double antibody sandwich (DAS)-ELISA test (Loewe Biochemica, Sauerlach, Germany), WSSM was detected serologically in all 15 collected wheat samples as well as in five wheat bait plants. For further confirmation, total RNA was extracted from leaves of all naturally infected and wheat bait plants using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and served as a template in reverse transcription (RT)-PCR. RT-PCR was carried out with One-Step RT-PCR Kit (Qiagen) using degenerate primers, WMVCPF and WMVCPR (Clover and Henry 1999), yielding an 879- to 882-bp fragment corresponding to the coat protein (CP) gene of both WSSMV and Wheat yellow mosaic virus (WYMV). Total RNAs extracted from healthy wheat leaves as well as RNase-free water were included as negative controls in RT-PCR analysis. Amplicons of the expected size were obtained from all 15 naturally infected and five bait wheat plants, while no amplification products were observed in the healthy controls. After the purification with QIAquick PCR Purification Kit (Qiagen), the RT-PCR product obtained from one selected isolate 361-13 was sequenced directly in both directions using the same primer pair as in RT-PCR (GenBank Accession No. KP257576). Pairwise comparison of the 361-13 isolate CP sequence with other homologous sequences available in GenBank, conducted using MEGA5 software (Tamura et al. 2011), revealed that wheat isolate from Croatia showed the highest nucleotide identity of 99.2% (100% amino acid identity) with the WSSMV isolate (AJ237926) originating from the United States. To our knowledge, this is the first report of WSSMV occurrence on wheat in Croatia. Wheat is the most important field crop in Croatia and the presence of this harmful virus could represent a major threat to its production. Further investigation toward establishing distribution of WSSMV and P. graminis as its vector will be conducted, followed by testing the resistance of local cultivars.