P HYTOPATHOLOGY / M YCOLOGY K. Erzurum et al.(2005) Phytoparasitica 33(3):261-266 Passalora Blight of Anise (Pimpinella anisum) and Its Control in Turkey K. Erzurum,1 F. Demirci,1 A. Karakaya,1 E. Çakır,2 G. Tuncer2 and S. Maden1,∗ Passalora blight of anise, caused by Passalora malkoffii (Bubák) U. Braun, is an important disease of anise in Turkey. The disease affects all the aboveground parts of plants including flower clusters. Infected seeds have dark, linear stromata. Detection of the pathogen on seeds was studied by the blotter method, agar method, washing test and sowing infected seeds in disease-free soils. The pathogen was recovered only by the washing test and to a limited extent by water agar + seed decoction agar. Sixteen of 24 seed samples from diseased regions were found to be infected. The pathogen was not detected by any other methods. However, several indigenous fungi, e.g. Alternaria alternata, were isolated, which may have prevented the growth of the pathogen. Seed washings of infected seed samples had typical spores of the pathogen up to 106 conidia per gram of seed. Transmission of the pathogen was shown by sowing infected seeds in disease-free field soils in two locations where anise had not been grown previously. Azoxystrobin, chlorothalonil + carbendazim and flutriafol seed treatments at 0.04 g a.i. kg−1 seed, 1.0 g + 4.5 g a.i. kg−1 seed and 0.015 g a.i. kg−1 seed reduced the disease by 92.5%, 89.6% and 36.2% in 2002 and by 78.9%, 75.8% and 41.2% in 2003, respectively. Three foliar applications of azoxystrobin, chlorothalonil + carbendazim and flutriafol at the rates of 187.5 g a.i. ha−1 , 1500 g + 6750 g a.i. ha−1 and 31.3 g a.i ha−1 reduced disease incidence by 92.5%, 86.0% and 96.8% in 2002 and by 97.5%, 90.8% and 97.0% in 2003, respectively. KEY WORDS: Anise; Passalora malkoffii; control; Turkey. INTRODUCTION Anise (Pimpinella anisum L.) is a member of the Umbelliferae and widely grown in the Mediterranean and Aegean regions of Turkey, namely, in Antalya, Burdur and Denizli provinces. It is used primarily in the production of raki, an alcoholic drink, and 80% of the total production (22,067 tons on 35,000 ha) of the country comes from this region (2). Blight of anise caused by Passalora malkoffii (Bubák) U. Braun was first described in 1906 from samples collected at Sadovo, near Philippopel, Bulgaria, as Cercospora malkoffii (5). Later on, a disease under the same name was reported from other countries where anise was grown (7,8,10,12). In Turkey, it was first mentioned by Bremer (4), who gave a brief description of the causal agent and its symptoms. The generic name of the pathogen recently has been changed to Passalora (3,6). The diseased samples collected from Turkey were also identified as Passalora malkoffii by U. Braun and they were kept Received Oct. 15, 2004; accepted Jan. 30, 2005; http://www.phytoparasitica.org posting May 17, 2005. 1 Dept. of Plant Protection, Faculty of Agriculture, Ankara University, 06110 Dışkapı, Ankara, Turkey. *Corresponding author [Fax: +90-312-3187-029; e-mail: maden@agri.ankara.edu.tr]. 2 Plant Protection Central Research Institute, Yenimahalle, Ankara, Turkey. Phytoparasitica 33:3, 2005 261 in the Martin Luther Universität Inst. f. Geobotanik und Botanischer Garten Herbarium (collection #HAL 1750). A serious disease epidemic occurred in 2000 in the anise-growing region of Turkey and the estimated annual crop loss due to this disease in this region is approximately $10 million (9). The disease is widespread and damaging in some warm and rainy years, infecting mostly seed clusters. Seed transmission of the pathogen and its control have not been studied. The present study deals with detection of the pathogen on the seeds, transmission of the disease and its control. MATERIALS AND METHODS Detection of P. malkoffii on seeds Various methods were employed to detect the pathogen on the seeds. Seeds were examined under a stereomicroscope for morphological variations. Seed samples showing black discolorations were washed by shaking 5 g seeds in 20 ml water for 2 minutes and the suspension was examined under a microscope for the presence of the spores of the pathogen. This test was conducted for all 24 seed samples. Seeds of a sample having abundant spores were incubated on moistened blotter papers with and without pretreatment by 0.1% NaOCl for 3 min; on agar media such as potato dextrose agar (PDA), half-strength PDA amended with seed decoction, Cercospora Selective Medium of Stavely and Nimmo (13), V8 juice agar and water agar (WA) alone and amended with seed decoction with pretreatment. The seed decoction was prepared by boiling 10 g of anise seeds in 100 ml of water for 2 min and 500 ml extract was then added to 1 l of the respective agar medium. Incubation of all the applications was done under a 12L:12D regime, with light provided by two 40 W near-ultra-violet tubes at 20±1 ◦ C. In another study, seeds were kept at 4◦ C for 10 days to break the possible dormancy of the fungus and incubated on WA with and without seed extract, half-strength PDA with seed extract and their controls under the same incubation conditions. One hundred seeds (20 seeds per 10-cm-diam petri dish) with four replications, all together 400 seeds, were used in all the applications. In all the agar media incubation studies, pretreated seeds were used. Seed transmission Seed transmission of the pathogen was investigated by sowing 100 seeds from a discolored seed sample in four replications in a sterile soil (sterilized at 121 ◦ C for 1 h on two consecutive days) mixture of peat, sand and field soil (1:1:2, v:v:v). The experiment was carried out in controlled climate rooms with a 12-h photoperiod illuminated by fluorescent lamps of 0.17 W per sec at 22±2◦ C. Seeds were also sown in two fields in which anise had not been previously grown and which were 300 km distant from anisegrowing areas of Turkey. The soils in the experiment fields were loamy in texture. The pots and the plots in the field were watered as needed. A clean seed sample which was determined by the washing test was also included in the experiment. Aerial spore dispersal to the clean plots was determined by Burkard spore traps (Burkard Manufacturing Co. Ltd., Rickmansworth, Herts., UK) during the entire growing season. Fungicide treatments Azoxystrobin (QuadrisSC 250), carbendazim + chlorothalonil (Takistin50% WP + Hektanil50% WP) and flutriafol (Impact125 SC) were evaluated as seed treatment. They were applied as a slurry at the rates of 0.04 g a.i. kg −1 seed, 1 g + 4.5 g a.i. kg−1 seed and 0.015 g a.i. kg−1 seed, respectively. Twenty g of seeds were used for each plot. Treated seeds were mixed with sand (1:3, v:v) and then immediately sown in the test plots (1.5×4.5 m) by spreading the mixture evenly on the plots and incorporated into 262 K. Erzurum et al. the soil to a depth of 2–3 cm with the help of a rake, according to usual farming practice. Foliar treatments with azoxystrobin, carbendazim + chlorothalonil and flutriafol were first applied at the 5–6-leaf stage at rates of 187.5 g a.i. ha−1 , 1500 g + 6750 g a.i. ha−1 and 31.3 g a.i. ha−1 in 2500 l ha−1 of water. The second and third applications were given at 15-day intervals. Treatments were applied with a knapsack pulverizer under 3 kg cm−2 pressure. Field experiments were done at the Haymana Experimental Farm of the Agricultural Faculty of Ankara University during the years 2002 and 2003. All the fungicide treatment experiments were conducted in a completely randomized block design with four replications and repeated in two consecutive years. There was an average of 200±20 plants in each plot and they completely covered the whole plot at the flowering stage. Disease estimation Disease severity was determined as mean percent disease at flowering stage by visual observations on 150 plants collected from five points from each of the plots. MINITAB (Minitab Inc., State College, PA, USA) and MSTAT (Michigan State Univ., East Lansing, MI, USA) package programs were used in the statistical analyses. RESULTS Disease symptoms and the pathogen The pathogen caused blight on all aboveground parts of the plants. Leaf blight symptoms started from the edges of leaflets as a sectorial necrosis (Fig. 1a). Abundant conidia and conidiophores were observed, especially on the lower surfaces of the leaves, as a velvety cover. The conidiophores were very short (5– 25×4–7 µm) and arranged in dense sporodochial conidiomata (Fig. 1b). Conidia were subcylindric-obclavate, fusiform, 26.3–107.5×2.5–5.73 µm (average 53×4 µm), hyaline, subhyaline to very pale yellowish green or olivaceous, mostly 1–3 septate (Fig. 1c). TABLE 1. Average disease severity and inhibition of disease development on anise grown from treated seeds at Haymana experimental plots in 2002 and 2003 z Fungicides 2002 Rate (g a.i. kg−1 seed) Disease severity (%) 76.9±7.46ay 5.7±089b Inhibition (%) 2003 Disease severity (%) 16.4±9.58a 3.5±1.16b Inhibition (%) Control Azoxystrobin 0.04 92.5 78.9 Carbendazim + chlorothalonil 1 + 4.5 8.0±3.96b 89.6 4.0±2.38b 75.8 Flutriafol 0.015 49.0±29.55a 36.2 9.50±3.76ab 42.2 z The values were calculated from averages of four replications; values for each year were analyzed separately. y For each year, values followed by a common letter do not differ significantly (P=0.05) according to Duncan’s Multiple Range Test. Detection of the pathogen on seeds Examination of seed samples revealed that infected seeds were darker than the healthy ones (Fig. 1d) due to black stromata of the pathogen (Fig. 1e). Stromata appeared as raised, linear dark areas. There were typical conidia of P. malkoffii in the suspension of seed washings of the dark seed samples. The numbers of conidia of the infected seed samples ranged from 103 to 106 per gram of seeds. Sixteen of Phytoparasitica 33:3, 2005 263 24 seed samples collected from diseased locations were found to have dark stromata in dry examinations and conidia in seed washings. Fig. 1. Various aspects of Passalora blight, the disease and the causal agent: (a) Disease symptoms on anise leaves; (b) conidiophores of Passalora malkoffii; (c) conidia of P. malkoffii; (d) healthy and (e) diseased anise seeds. TABLE 2. Average disease severity and inhibition of disease development on anise at Haymana experimental plots after three foliar applications in 2002 and 2003 z Fungicides Control Azoxystrobin Carbendazim + chlorothalonil Rate (g a.i. ha−1 ) 187.5 2002 Disease severity (%) 49.1±3.68ay 3.7±1.77c Inhibition (%) 92.5 2003 Disease severity (%) 21.4±11.94a 0.5±0.38b Inhibition (%) 97.5 1500 6.9±3.34b 86.0 0.5±0.22b 97.8 +6750 Flutriafol 31.3 1.6±0.29c 96.8 0.6±0.27b 97.0 z The values were calculated from averages of four replications; values for each year were analyzed separately. y For each year, values followed by the same letter do not differ significantly (P=0.05) according to Duncan’s Multiple Range Test. 264 K. Erzurum et al. In the blotter test most of the seeds, surface disinfested or not, were covered with non-pathogenic fungi, especially Alternaria alternata. These fungi probably prevented the detection of P. malkoffii. None of the tested media yielded fructifications of the fungus except WA amended with a seed decoction, where growth of P. malkoffii was observed on three seeds out of 200. Transmission of the pathogen by seeds No clear symptom on the plants grown from infected seeds in controlled growth rooms was obtained. Some plants had red discolorations on leaves but no fungal growth was observed. When infected seeds were sown in the field at two locations which had never been sown with anise, very profuse disease development and fungal sporulation were seen. Disease severity reached approximately 90% at the end of the season. There were no disease symptoms in the control plots nor any spore spread to them. Control of the disease Seed treatments: Azoxystrobin and carbendazim + chlorothalonil seed treatment effectively reduced seed transmission of the pathogen in the field (Table 1). Azoxystrobin reduced the disease by 92.5% and 78.9% in 2001 and 2002, respectively. The reduction for carbendazim + chlorothalonil was 89.6% and 75.8% for 2001 and 2002, respectively. Flutriafol seed treatment was not very effective, achieving only 36.2% and 42.2% control in 2001 and 2002. Foliar treatments: When diseased seeds were sown, uniform disease symptoms appeared in the control plots. All the foliar fungicides controlled the disease (Table 2) in both years. The effectiveness of carbendazim + chlorothalonil in 2002 was slightly lower than that of the other fungicides. DISCUSSION The pathogen was identified on the basis of the description given by Braun and Melnik (3). The conidia of P. malkoffii described by these authors are (0-)1-3 (-5)-septate, hyaline, subhyaline to very pale yellowish green or olivaceous, subcylindric-obclavate, fusiform, (20-) 30-60 (-90) × (2-) 3-5 (-6) µm (minimum, average and maximum values). The septation and the form of the conidia in our observations were in close agreement with these authors. Conidiophore structure and measurements were also similar. Even though the disease has been reported from several countries, seed transmission has not been demonstrated. Our field studies concerning seed transmission were conducted at two locations in Ankara (field crops experimental area at Dışkapı, and at the Haymana Experimental Farm of Ankara University). No anise has been grown in these areas and the closest anise growing area is about 300 km away. The high rate of disease (76.0%) reached in these disease-free areas by sowing infected seeds shows the importance of disease transmission. Visual examination and washing the seeds were useful in detection of the pathogen on seeds. Transmission of P. malkoffii was not evident in controlled studies with sterile soil, and the reason for this should also be investigated. There are many different effects of media and incubation conditions on sporulation and growth of cercosporoid species (1,11,13,14) and the species P. malkoffii did not grow and sporulate on any of the media tested. Further work is needed to determine the growth and sporulation of this species on various media with different incubation conditions. It appears that there are some factors affecting seed transmission in the field and these do not exist under controlled conditions. Among these factors are temperature fluctuations of up to Phytoparasitica 33:3, 2005 265 15–20◦ C between day and night, day length, and the quality and intensity of the light. Soil factors are probably not so important since we obtained the disease with different soil types at different locations. ACKNOWLEDGMENTS The authors are grateful to the Research Fund of Ankara University for supporting this work, which is part of project No. 2000.07.11.009; and to Uwe Braun from Martin-Luther Universität, F.B. 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