Berna Tunali

Root and Crown Rot Fungi Associated with Spring, Facultative, and Winter Wheat in Turkey

The objective of this study was to determine the distribution frequency of the fungi associated with wheat (Triticum aestivum) crowns and roots in cereal producing areas of Turkey through a targeted survey of 518 commercial fields over a 2-year period. More than 26% of the fields had one or more of the fungal species commonly reported as part of the dryland root rot complex, Fusarium culmorum (14%) > Bipolaris sorokiniana (10%) > F. pseudograminearum (2%). The fungi considered to be part of the high rainfall root rot complex were found at very low frequencies: 2% for Gaeumannomyces graminis and 3% for Pythium spp. Species of Rhizoctonia were found in 22% of the fields. Several Fusarium species considered to be less or nonpathogenic to cereals were also found in high frequencies at 11% (F. oxysporum, F. chlamydosporum), 10% (F. sporotrichioides), and 8% (F. avenaceum and F. solani). The mostly random distribution of cereal root-rotting species across the survey area suggests the fungi are not distributed in any distinct agroecological relationship. As a result, the relative economic importance of a given species on wheat will be determined by a number of factors, such as their fungal pathogenicity, host susceptibility/tolerance, and the seasonal conditions. Results from this study suggest that there are a wide range of fungal species associated with root and crown tissues of wheat.

Fusarium culmorum is a single phylogenetic species based on multilocus sequence analysis

Fusarium culmorum is a major pathogen of wheat and barley causing head blight and crown rot in cooler temperate climates of Australia, Europe, West Asia and North Africa. To better understand its evolutionary history we partially sequenced single copy nuclear genes encoding translation elongation factor 1-α (TEF), reductase (RED) and phosphate permease (PHO) in 100 F. culmorum isolates with 11 isolates of Fusarium crookwellense, Fusarium graminearum and Fusarium pseudograminearum. Phylogenetic analysis of multilocus sequence (MLS) data using Bayesian inference and maximum parsimony analysis showed that F. culmorum from wheat is a single phylogenetic species with no significant linkage disequilibrium and little or no lineage development along geographic origin. Both MLS and TEF and RED gene sequence analysis separated the four Fusarium species used and delineated three to four groups within the F. culmorum clade. But the PHO gene could not completely resolve isolates into their respective species. Fixation index and gene flow suggest significant genetic exchange between the isolates from distant geographic regions. A lack of strong lineage structure despite the geographic separation of the three collections indicates a frequently recombining species and/or widespread distribution of genotypes due to international trade, tourism and long-range dispersal of macroconidia. Moreover, the two mating type genes were present in equal proportion among the F. culmorum collection used in this study, leaving open the possibility of sexual reproduction.

Fitness of three Fusarium pathogens of wheat.

Crown rot and head blight of wheat are caused by the same Fusarium species. To better understand their biology, this study has compared 30 isolates of the three dominant species using 13 pathogenic and saprophytic fitness measures including aggressiveness for the two diseases, saprophytic growth and fecundity and deoxynivalenol (DON) production from saprophytic colonization of grain and straw. Pathogenic fitness was generally linked to DON production in infected tissue. The superior crown rot fitness of Fusarium pseudograminearum was linked to high DON production in the stem base tissue, while Fusarium culmorum and Fusarium graminearum had superior head blight fitness with high DON production in grains. Within each species, some isolates had similar aggressiveness for both diseases but differed in DON production in infected tissue to indicate that more than one mechanism controlled aggressiveness. All three species produced more DON when infecting living host tissue compared with saprophytic colonization of grain or straw, but there were significant links between these saprophytic fitness components and aggressiveness. As necrotrophic pathogens spend a part of their life cycle on dead organic matter, saprophytic fitness is an important component of their overall fitness. Any management strategy must target weaknesses in both pathogenic fitness and saprophytic fitness.

Genetic diversity and mating types of Fusarium culmorum and Fusarium graminearum originating from different agro-ecological regions in Turkey

Fusarium culmorum and F. graminearum are the major pathogens for dryland root/foot‐rot and head‐blight diseases in economically important grain crops. This study was aimed at the molecular characterization of Fusarium spp. isolates, which have been collected from cereal fields in three agro‐ecological regions in Turkey. Genetic diversity has been analyzed by generating RFLP markers from the intergenic spacer (IGS) region of ribosomal RNA. The selection of restriction enzymes for IGS‐RFLP studies has been found critical to maximize polymorphic markers. Only 3 of 14 restriction endonucleases were useful in differentiating Fusarium spp. isolates. PstI was the most efficient enzyme to produce a maximum of nine DNA markers in one individual and total 22 polymorphic representative banding patterns. Polymorphism based on IGS‐RFLP was high and average 88% in both species. There was no association between IGS diversity and geographic locations from which the samples were taken. Both MAT‐1 and MAT‐2 sequences were amplified in F. graminearum similarly to previous reports. Most of the F. culmorum isolates carried either MAT‐1 or MAT‐2 sequences, and differently two isolates carried both sequences. Mating type determination was helpful to distinguish F. pseudograminearum from F. graminearum, which cannot be discriminated by SCAR markers or morphological assessment. High genetic diversity by IGS‐RFLP markers in F. culmorum was discussed in relation to its fitness as the most common pathogen in dryland root rot complex (DLRRC).

First report of Cylindrocarpon liriodendri on kiwifruit in Turkey.

During a routine survey of diseases of kiwifruit (Actinidia chinensis Planch.) cv. Hayward conducted in autumn of 2009 in Ardeşen, Rize Province (eastern Black Sea Region, Turkey), symptoms of a new disease were observed in five locations. Affected trees showed leaf wilting that frequently led to the death of the trees. Symptoms at ground level included necrotic lesions on woody tissues of both the rootstock and roots. Small pieces from necrotic wood and root tissues were surface disinfested and plated onto potato dextrose agar (PDA) medium amended with 0.5 g liter-1 of streptomycin sulfate and incubated for 7 days at 25°C in the dark. Isolates were transferred to PDA and presumptively identified as a Cylindrocarpon sp. by morphology and conidial characteristics. The isolates were transferred to PDA and Spezieller Nährstoffarmer Agar (SNA) and then incubated at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed floccose to felted mycelium, which varied in color from brown-yellow to sepia. On SNA, all isolates produced microconidia measuring 6.25 to 15 (9.6) × 2.5 to 5 (3.02) μm and macroconidia of one-septate measuring 7.5 to 20 (13.3) × 2.5 to 5 (3.8) μm, two-septate measuring 12.5 to 25 (20.7) × 3.25 to 5 (4.58) μm, and three-septate measuring 16.3 to 30 (11.04) × 3.75 to 5 (4.82) μm. Chlamydospores 7.5 to 11.3 (9.78) μm were intercalary or terminal in the mycelium, single or occasionally in chains. Identity of these isolates was determined by a multiplex PCR system using a set of three pairs of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a product size of 253 bp, which is characteristic of Cylindrocarpon liriodendri J.D. MacDonald and E.E. Butler, in agreement with morphological features (2). Additionally, the internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolates 10K-TR1 and 10K-TR2 and deposited in GenBank (Accession Nos. HQ113122 and HQ113123). These sequences showed high similarity (98%) with the sequence of C. liriodendri (GenBank Accession No. DQ718166). A pathogenicity test was conducted using isolate 10K-TR1 and repeated twice. Six 8-month-old callused and rooted cuttings of kiwifruit cv. Hayward were surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, washed twice with sterile distilled water (SDW), and inoculated by dipping their roots for 30 min in a spore suspension of the fungus (1 × 106 conidia ml-1) obtained from 30-day-old colonies grown on PDA. Six control cuttings were dipped in SDW. Two weeks later, cuttings were drench inoculated with 50 ml of the designated spore suspension to guarantee root infection and controls were drenched again with SDW. Plants were maintained in a greenhouse with a temperature range of 25 to 30°C. Four months after inoculation, the inoculated plants developed wilting and root symptoms similar to those observed in natural infections and C. liriodendri was reisolated, completing successfully Kochs postulates. No symptoms were observed on the control plants. To our knowledge, this is the first report of C. liriodendri on kiwifruit trees in Turkey. References: (1) S. Alaniz et al. Plant Dis. 93:821, 2009. (2) F. Halleen et al. Stud. Mycol. 55:227, 2006.

First report of rust disease caused by Uromyces galegae on Galega officinalis in Turkey.

Galega officinalis L. is an obnoxious invasive weed in the United States and a potential target for biological control efforts. The plant, a member of the legume family, is native to western Asia and southern Europe. During September 2001, uredinial pustules were observed on leaves of G. officinalis L. in Kizilcahamam, Ankara. Specimens were examined microscopically and compared with published descriptions (2) and herbarium specimens in the U.S. National Fungus Collections, Beltsville, MD. The fungus was subsequently identified as Uromyces galegae (Opiz) Sacc. on the basis of morphological characteristics of the uredinia, urediniospores, and teliospores. The following description is from the Turkish material: uredinia subcircular to oblong, hypophyllous, rarely epiphyllous at petiole, and 0.5 to 1 mm in diameter; urediniospores subovoid to subglobose, 17.5 to 20.0 × 19.5 to 22.5 μm (average = 18.0 × 20.0 μm), wall 1 to 2 μm thick, finely echinulate, cinnamon brown, and with 3 to 5 usually equatorial germ pores; telia similar to uredinia; teliospores irregular in shape ranging from globose to ovoid to triangular, apex papillate, wall 2 to 3 μm thick, thicker at the apex, chestnut brown, strongly verrucose to tuberculate, 17.5 to 22.5 × 22.5 to 27.5 μm (average = 20.3 × 24.5 μm), pedicel hyaline, and easily broken. Voucher specimens are deposited in the U.S. National Fungus Collections (BPI 863535); a nucleotide sequence spanning the ITS2 and 28S rDNA genes of this isolate was obtained and deposited in Gen-Bank (Accession No. DQ250133). U. galegae has been reported on G. officinalis from Bulgaria, Greece, and Italy (1). To our knowledge, this is the first report of U. galegae in Turkey and marks the eastern-most record for its distribution. References: (1) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory, On-line publication, USDA-ARS, 2005. (2) M. Pantidou and D. M. Henderson. Notes R. Bot. Gard. Edinb. 29:277, 1969.

FIRST REPORT OF LEAF SPOT OF CONVOLVULUS ARVENSIS CAUSED BY PHOMA MACROSTOMA var. MACROSTOMA IN TURKEY

In May 2012, Convolvulus arvensis plants with irregular tan-colored necrotic lesions on the leaves were observed near Ondokuz Mayis University (Samsun, Turkey). Leaves with lesions were surface-disinfected for 5 min in 10% bleach followed by incubation at 20-25°C in a moist chamber. Pycnidia developed after 5-7 days, and pure cultures were obtained by transferring conidia from pycnidia onto oatmeal agar. Colonies were peach-colored, and did not change color with the addition of NaOH. Pycnidia in culture were scattered, globose, 119.6 μm in diameter, with one papillate ostiole (average 29.7 μm in diameter) with glabrous and elongated necks (average 199.5 μm in length). Conidia were aseptate, hyaline, and ellipsoidal to oblong (average 8.1×2.7 μm). Conidial matrix color varied from gray to flesh to salmon. These characters matched the description of Phoma macrostoma Mont. var. macrostoma (Boerema et al., 2004). A specimen was deposited with the US National Fungus Collections (BPI 892537). DNA sequences for the internal transcribed spacers (GenBank accession No. KC590613) aligned, after BLAST analysis, 97% to 10 isolates of P. macrostoma var. macrostoma and 99% to two isolates of P. macrostoma (GenBank Accession Nos HM755951 and DQ474091). Conidia were spray-inoculated, in a suspension of 106 conidia ml-1, onto twenty 30-day-old C. arvensis plants; another 10 control plants were sprayed with water only. Plants were placed in a dew chamber at 25°C for 40 h, then moved to a 20-25°C greenhouse. All inoculated plants but none of the controls, became diseased. P. m. var. macrostoma was re-isolated from all inoculated plants. To our knowledge this is the first report of P. m. macrostoma from C. arvensis.

First Report of Anthracnose of Mile-a-Minute (Persicaria perfoliata) Caused by Colletotrichum cf. gloeosporioides in Turkey

Mile-a-minute (Persicaria perfoliata (L.) H. Gross; family: Polygonaceae) is an exotic annual barbed vine that has invaded the northeastern USA and Oregon (2). In July of 2010, in a search for potential biological control pathogens (3), diseased P. perfoliata plants were found along the Firtina River near Ardesen, Turkey. Symptoms were irregular dark necrotic lesions along leaf margins and smaller irregular reddish lesions on the lamellae of leaves. Symptomatic leaves were sent to the quarantine facility of FDWSRU, USDA, ARS in Ft. Detrick, MD, for pathogen isolation and testing. Symptomatic leaves were excised, surface disinfested in 0.615% NaOCl, and then incubated for 2 to 3 days in sterile moist chambers at 20 to 25°C. Numerous waxy sub-epidermal acervuli with 84-μm-long (mean) black setae were observed in all of the lesions after 2 to 3 days of incubation. Conidiophores within acervuli were simple, short, and erect. Conidia were one-celled, hyaline, guttulate, subcylindrical, straight, 12.3 to 18.9 × 3.0 to 4.6 μm (mean 14.3 × 3.7 μm). Pure cultures were obtained by transferring conidia onto 20% V-8 juice agar. Appressoria, formed 24 h after placing conidia on dialysis membrane over V-8 juice agar, were smooth, clavate, aseptate, regular in outline, and 6.4 to 10.0 × 5.1 to 7.2 μm (mean 7.5 × 6.6 μm). These characters conformed to the description of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (1). A voucher specimen was deposited in the U.S. National Fungus Collections (BPI 882461). Nucleotide sequences for the internal transcribed spacers (ITS 1 and 2), directly sequenced from ITS 1 and ITS 4 standard primers (4), were deposited in GenBank (JN887693). A comparison of these sequences with ITS 1 and 2 sequences of the C. gloeosporioides epitype IMI 356878 (GenBank EU 371022) (1) using BLAST found 479 of 482 identities with no gaps. Conidia from 14-day-old cultures, in an aqueous suspension of 1.0 × 106 conidia ml-1, were spray-inoculated onto healthy stems and leaves of twenty 30-day-old P. perfoliata plants. Another 10 plants were not inoculated. All plants were placed in a dew chamber at 25°C for 16 h with no lighting. They were then placed in a 20 to 25°C greenhouse with a 14-h photoperiod. Light was generated using 400W sodium vapor lights. Lesions developed on leaves and stems of all inoculated plants after 7 days, and symptoms were the same as observed in the field. Each plant was rated weekly for disease severity on a 0 to 10 rating scale where 0 = no disease symptoms and 10 = 100% symptomatic tissue. After 28 days, the average disease rating of inoculated plants was 3.95 ± 0.94. No disease developed on noninoculated plants. C. gloeosporioides was reisolated from all inoculated plants. Host range tests will determine the potential of this isolate as a biological control agent for P. perfoliata. To our knowledge, this is the first report of anthracnose caused by C. gloeosporioides on P. perfoliata. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) J. T. Kartesz and C. A. Meacham. Synthesis of the North American Flora, Version 1.0., North Carolina Botanical Garden, Chapel Hill, N.C. 1999. (3) D. L. Price et al. Environ. Entomol. 32:229, 2003. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Evaluation of Trichoderma spp. from central and northern regions of Turkey for suppression of Polymyxa betae as a vector of rhizomania disease.

In the current study, 18 Trichoderma spp. isolates were obtained from different provinces in central and northern regions of Turkey. The ability of nine selected isolates to suppress the colonisation of roots by P. betae and the multiplication of BNYVV in sugar beet roots under controlled conditions were tested. Roots of seedlings growing in the P. betae-BNYVV-infested soil were analysed by enzyme-linked immunosorbent assay to test for the presence of BNYVV and checked microscopically for the density of cystosori of P. betae. The numbers of P. betae resting spores in cystosori for each treatment were counted using a light microscope. Except for isolates Tr-1 and Tr-5, the effect of selected Trichoderma isolates on suppressing multiplication of BNYVV varied between 4 and 53%. The total number of resting spores in the roots varied between 14.4 and 25.1 for the different Trichoderma spp. treatments. The lowest number of resting spores in clusters was recorded in T. harzianum Tr-8. In addition, the shapes of resting spores were not normal in the Tr-8 treatments. The cystosori from this treatment were also abnormally dark in colour and had deformed walls.