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Journal of Plant Pathology (2009), 91 (2), 299-302 Edizioni ETS Pisa, 2009 299

VIABILITY OF GRAMINEA CULTURES AFTER SUNLIGHT EXPOSURE UNDER FIELD CONDITIONS

M.I.E Arabi, E. Al-Shehadah and M. Jawhar

Department of Molecular Biology and Biotechnology, AECS, P O Box 6091, Damascus, Syria

SUMMARY linked with the arrival of more aggressive genotypes of the causal agent (Arabi and Jawhar, 2007), thus calling Conidia of Pyrenophora graminea, the causal agent of for renewed and improved disease management. In ad- leaf stripe, were simultaneously exposed out- dition to the differential levels of pathogenicity of di- doors to direct solar radiation or placed in an adjacent verse P. graminea strains, variation in the occurrence of ventilated enclosure in the dark for four months. After the pathogen linked with changes in weather conditions exposure, conidia were placed on water agar in closed has contributed significantly to the sporadic nature of Petri dishes and allowed to germinate for 24 h. Shaded BLS. In the 1990’s, P. graminea was not detectable in conditions were always more favourable to conidia ger- many Syrian locations. Clearly, forecasts could be im- mination and mycelial growth than sunlight conditions. proved if they comprised not only information on the Significant decreases (P<0.05) in conidia germinability presence/absence of the pathogen in an area, but also (60.6%) and mycelial growth (41.46%) were detected the risks associated with varying levels of inoculum. in light-exposed conidia in comparison with the non-ex- Since our long-term goal is to identify the factors that posed control. Exposure also decreased the pathogenic- favour BLS development, the aerial dispersal of fungal ity of on different cultivars. The possibility that spores was investigated, being a likely determinant of sunlight exposure may reduce conidia germinability disease outbreaks. To this aim, the effects of sunlight on over the hottest four months and aerial transport dis- conidial survival under field conditions was studied, for tances should be considered. these effects are likely to be important in determining P. graminea survival of during dispersal. Key words: Pyrenophora graminea, barley, leaf stripe, spore survival, solar radiation. MATERIALS AND METHODS

INTRODUCTION Experimental material. The isolate of P. graminea Sy3 used in this study was chosen among 15 isolates, as the Pyrenophora graminea Ito & Kuribayashi [anamorph most virulent isolate on barley differential genotypes graminea (Rabenh. ex. Schlech. Shoem.)] is (Arabi et al., 2005). At the end of the growing season, the seed-borne agent of barley leaf stripe (BLS), a dis- conidia of Sy3 were produced on the leaf surface of bar- ease responsible for a 73% reduction of annual yield in ley (cvs WI 2291, Golf and Igri) by wetting plants twice highly susceptible barley varieties in Syria (Arabi et al., a day, using a high-pressure sprayer. 2004). Aerial dispersal of this fungus is considered to be an Exposure to sunlight. The exposure of conidia to sun- important factor in the spreading of BLS epidemics light was performed between June to October 2006 un- (Davis and Jackson, 2005). When conditions are wet or der field conditions approximately 22 km west Damas- humid, spores are produced on the leaf surface at about cus. Two sets of samples were used, kept under direct the time when spikes of healthy plants in the field begin sunlight and in the dark. After harvesting, plants infected to flower. Spores are dispersed by wind to developing with Sy3 were exposed to full sunlight under field condi- spikes, germinate, and cause infections. tions and four leaves were taken from each plant at the The recent resurgence of BLS in Syria appeared to be end of exposure for further experiments. Sy3 conidia in the second set were transferred to glass microscope-slide coverslips (18 mm × 18 mm × 0.14 mm) by touching lightly a BLS lesion with a coverslip. Conidia-laden cov- Corresponding author: M.I.E. Arabi Fax: +963.11.612289 erslips were then placed on screens in a darkened enclo- E-mail: [email protected] sure with ventilation. The experiment consisted of 10 003_JPP324Arabi_299 25-06-2009 10:44 Pagina 300

300 Sunlight effect on Pyrenophora graminea viability Journal of Plant Pathology (2009), 91 (2), 299-302

coverslips (six replicates) for each treatment. rainfed conditions (500 mm annual rainfall) in a com- pletely randomized block design, with three replicates Meteorological data. Global solar irradiance (watts (50 plant/replicate/cultivar). Plots were 1 × 1 m in size per square meter), air temperature, relative humidity with a 1 m buffer. Each replicate consisted of five rows, (RH) and wind speed were measured during the experi- 25 cm apart with 10 seeds sown per row. At the heading ment using a data logger (model 1020, COMBILOG, stage (GS 50) diseased (showing leaf stripe) and healthy Theodor Friendrichs & Co. Hamburg, Germany). Air plants were counted. The degree of resistance to BLS temperature and RH were measured with a platinum re- was assessed as the percentage of infected plants ac- sistance thermometer sensor (pt100, DIN 60751 B) and cording to Delogu et al. (1989). wind speed with a cup anemometer (model 014A, COMBILOG, Germany). These instruments were lo- Measure of mycelial growth. Mycelial growth was cated in the test site, at a height of 1.1 m above the measured at the end of sunlight exposure by culturing ground. The data were recorded at 1 h intervals. the exposed conidia on Petri dishes containing PDA with 13 mg/l kanamycin sulphate. The plates were incu- Germinability assessment. At the end of the experi- bated at 21±1ºC in the dark to allow fungal growth. ment sunlight-exposed and shaded conidia were collect- Comparisons with controls were made by measuring ed. Microscope coverslips were placed conidia-side down mycelial growth of three or four colonies per plate 8 on 1.5% water agar medium in 9-cm plastic Petri dishes days after culturing (six replicates). (6 Petri dishes per treatment), incubated at 20-22ºC for 24 h to allow germination. Percentages of germinated Statistical analysis. All experiments were performed conidia were counted in random fields at 100X with a three times. An F-test was used to determine if the three light microscope. A total of 200 to 400 conidia were ex- runs of each experiment were homogeneous and if the amined in six replicates (Petri dish/replicate), one repli- data could be pooled, which showed that this was possi- cate matching a replicate in the field, with the higher ble. Thus, all further analyses were conducted on number of conidia counted when germinability was low. pooled data. Statistical analysis was performed using the A conidium was considered germinated if the length of STAT-ITCF program (Anonymous, 1988). Analysis of the germ was greater than or equal to its length. variance (Newman-Keuls test) was used to test for dif- ferences among exposed and non exposed conidia. Pathogenicity tests. Pathogencity tests of all exposed Analysis of pathogenicity tests was done on the percent- and non-exposed conidial samples were done using age of infected plants, to determine the effect of sun- three barley cultivars (WI 2291, Golf and Igri), selected light exposure. for their different resistance levels to BLS and inoculat- ed with Sy3 as described by Hammouda (1986). One- hundred and fifty seeds of each cultivar were surface RESULTS AND DISCUSSION sterilized in 5% NaOCl for 5 min, washed several times in sterile deionized water, and left to dry between steril- Table 1 shows the range of environmental conditions ized filter papers. Seeds of each cultivar were trans- encountered during exposure of P. graminea conidia to ferred to Petri dishes (50 seed each) containing an ac- sunlight. Germinability of conidia decreased (60.6%) tively growing mycelium (8-day-old) cultured on potato significantly (P<0.05) after sunlight exposure in com- dextrose agar (PDA, DIFCO, USA) and incubated at parison with the non-exposed controls. In addition, ex- 6ºC for 14 days in the dark. After inoculation the seeds posure to sunlight significantly (P<0.05) decreased were removed carefully and planted in the field. (41.46%) mycelial growth 8 days after exposure in com- The location of the experiment was favorable for the parison with the non-exposed control (Table 2). There development of BLS. Inoculated seeds were sown under were significant differences (P<0.05) in pathogenicity

Table 1. Range of magnitude of environmental conditions en- Table 2. Percent germination and mycelium growth of P. countered during the exposure of P. graminea to sunlight un- graminea isolate Sy3 exposed (GS) and not exposed to sun- der field conditions. light (GNS) (six replicates).

Variable Range (Unit) Treatment % Germination Growth rate (cm) Outdoor air temperature 25-36 (°C) (24h of culture) (8 days of culture) Relative humidity 35-49 (%) GNS 87.5a ± 3.421 4.1a ± 0.132 Average wind speed at sample height 0.8-3.3 (m/s) GS 34.47b ± 7.96 2.4b ± 0.260 Irradiance of incident solar radiation 690-900 (w/m2) Means followed by different letters differ significantly at Conditions in the lab were stable (P<0.05) by Newman-keul’s test. 003_JPP324Arabi_299 25-06-2009 10:44 Pagina 301

Journal of Plant Pathology (2009), 91 (2), 299-302 Arabi et al. 301

Table 3. Leaf stripe disease incidence (%) of three barley cultivars, S: sunlight, NS: non exposed to sunlight (three replicates).

Disease incidence (%)x Cultivar Source Severity NS S WI2291 USA 98.8a ± 1.276 87.0b ± 2.861 Up to flag leaf Golf England 58.26a ± 0.721 44.25b ± 4.491 Lower three leaves Igri Germany 22.5a ± 2.271 19.45b ± 2.589 First leaf

Means followed by different letters are significantly different at (P<0.05) by Newman-Keul’s test. x The percentage of infected plants according to Delogu et al. (1989).

between exposed and non-exposed fungus (Table 3). oped to accommodate readily new information on the Survival of conidia is of paramount importance in the biology of the fungus and the susceptibility of barley build-up of P. graminea inoculum, as conidia transport- cultivars to BLS as it becomes available ed from infested residue by the wind or rain can infect healthy plants. Although there may have been differ- ences in the ability of conidia at different ages to germi- ACKNOWLEDGEMENTS nate, we used the germination of non-exposed conidia as a base-line to compensate for these differences. The authors thank the Director General of AECS The present study has shown that exposure to sun- and the Head of Biotechnology Department for their light decreases both germinability and pathogenicity of support. They also thank Ms. A. Shoib and Mr. M. Ra- P. graminea conidia, in agreement with the results ob- jah for their much appreciated help. Thanks also ex- tained by Arabi and Jawhar (2003) with Cochliobolus tended to Dr A. Al-Daoude for his critical readings of sativus, and by Hughes et al. (2003) with antarctic ter- the manuscript. restrial fungi. Rotem et al. (1985) and Gates (1980) at- tributed the inhibiting effect to the strongly increases amount of biological activity per unit of energy follow- REFERENCES ing exposure to shorter wavelength light (254 nm). Mo- hammed and El-Hassi (1986) suggested that conidial Anonymous, 1988. STAT-ITCF, Programme, MICROSTA, re- survival under sunlight may increase the production of alized by ECOSOFT, 2nd Ver. Institut Technique des Cere- secondary metabolites that may play a role in protection als et des Fourrages Paris, pp. 55. from solar irradiation. Swan (1974) reported that Arabi M.I.E., Jawhar M., 2003. Germinability of Cochliobolus melanin can offer an excellent protection against sun- sativus conidia exposed to solar radiation. Journal of Phy- light in many fungi. Conidia of P. graminea are dark topathology 151: 620-624. brown, thus these pigments might be one of the reasons Arabi M.I.E., Jawhar M., Al-Safadi B., MirAli N., 2004. Yield behind their tolerance of solar irradiation during the response of barley to leaf stripe (Pyrenophora graminea) under experimental conditions in southern Syria. Journal first periods of exposure. of Phytopathology 152: 519-523. To our knowledge, this is one of the few studies on Arabi M.I.E., Jawhar M., MirAli N., 2005. Storage protein the effects of sunlight on the viability of conidia and (hordein) patterns of barley-Pyrenophora graminea interac- aerial dispersal of a seed-borne pathogen. The sunlight tion. Seed Science and Technology 33: 409-418. apparently poses no strong crucial impediment to the Arabi M.I.E., Jawhar M., 2007. Heterogeneity in Pyrenophora survival of conidia of P. graminea under field conditions graminea as revealed by ITS-RFLP. Journal of Plant Pathol- during the four hottest months in Syria. Therefore, al- ogy 89: 391-395. though conidia of this pathogen are able to travel sever- Davis M., Jackson L.F., 2005. UC IPM Pest Management al kilometers in the atmosphere, decrease in their ger- Guidelines: Small Grains UC ANR Publication 3466. Dis- minability due to sunlight may be relevant when starting eases. Statewide IPM Program, Agriculture and Natural inoculum levels are low. However, aerial dispersal alone Resources, University of California. cannot explain the presence BLS in new areas due to Delogu G.A., Porta-Puglia A., Vannacci G., 1989. Resistance the fact that P. graminea is seed-borne (Zriba and of winter barley varieties subjected to natural of Pyrenopho- Harrabi, 1995). Biological information will help deter- ra graminea. Journal of Genetics and Breeding 43: 61-66. mining spore dispersal and infection probabilities when Gates D.M., 1980. Biophysical Ecology. Springer-Verlag, New regional levels of inoculum are sufficiently low, so as to York. postpone or forego chemical control. The present work Hughes K.A., Lawley B., Newsham K.K., 2003. Solar UV-B is part of an on-going project on the epidemiology of radiation inhibits the growth of antarctic terrestrial fungi. leaf stripe in Syria. A model framework will be devel- Applied and Environmental Microbiology 69: 1488-1491. 003_JPP324Arabi_299 25-06-2009 10:44 Pagina 302

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Received July 27, 2008 Accepted January 13, 2009