Epidemiological Investigations of Black Leaf Mold (Pseudocercospora fuligena (Roldan) Deighton) on Tomato (Solanum lycopersicum L.) under Protected Cultivation Der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades eines Doktors der Gartenbauwissenschaften - Dr. rer. hort. - genehmigte Dissertation von Zelalem Mersha Ayele (MSc.) geboren am 20.10.1972 in Melkawerer, Äthiopien 2008 Epidemiological Investigations of Black Leaf Mold (Pseudocercospora fuligena (Roldan) Deighton) on Tomato (Solanum lycopersicum L.) under Protected Cultivation Der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades eines Doktors der Gartenbauwissenschaften - Dr. rer. hort. - genehmigte Dissertation von Zelalem Mersha Ayele (MSc.) geboren am 20.10.1972 in Melkawerer, Äthiopien Angefertigt am Institut für Pflanzenkrankheiten und Pflanzenschutz Hannover, Januar 2008 Referent: Prof. Dr. Bernhard Hau Institut für Pflanzenkrankheiten und Pflanzenschutz Naturwissenschafliche Fakultät der Gottfried Wilhelm Leibniz Universität Hannover Herrenhäuser Straße 2, 30419 Hannover Koreferent: Prof. Dr. Hartmut Stützel Institut für Biologische Produktionssysteme Naturwissenschafliche Fakultät der Gottfried Wilhelm Leibniz Universität Hannover Herrenhäuser Straße 2, 30419 Hannover Tag der Promotion: 22 Februar, 2008 Abstract i ABSTRACT Macroscopic observations of symptoms and signs of the disease as well as isolation, culturing, inoculation (Koch’s postulate) and microscopic characterizations (morphological and molecular) of fungal structures from axenic cultures proved Pseudocercospora fuligena to be the causative agent of black leaf mold (BLM) in Thailand. Macroscopically, the appearance of indistinct effuse patches on tomato leaves, amphigenous fructification and prolific fuliginous appearance of sporulating lesions, predominantly on the abaxial side and as infection progresses on the adaxial side too, were some of the peculiar features of the disease. Microscopically, the basic fungal structures like conidial dimension and segments (11-128 µm in length x 3.5–9 µm in width, with up to 12 septations) as well as fascicles of conidiophores of P. fuligena were observed. Despite its slowness, P. fuligena grew on all the ten artificial growth media tested but improved growth and sporulation occurred on tomato oatmeal agar and carrot leaf decoction agar, both supplemented with CaCO3. Stomatal penetration and egress of P. fuligena were testified after light and scanning electron microscopy observations of cleared as well as intact leaves with the staining using aqueous acid fuchsin solution. Further conidiogenesis studies on cleared and stained leaves indicated prevalence of primary and secondary infection hyphae and exponential progression of blocked stomatal apertures. With this background information of the pathogen, further studies on determinants of the “infection chain” in terms of favorability of temperature (T), wetness duration (WD) and leaf age (A) were carried out under ambient (AE) and controlled environment (CE) experiments. P. fuligena under artificial inoculation in CE experiments was highly favored by a temperature of 28°C. Considering the high mean daily T that prevailed under AE experiments in greenhouses at central Thailand (duration of < 11 h at T ≤ 28°C and of about 17 hours at T ≤ 30°C), P. fuligena could be assumed to cause the disease even at a T that surpasses the limit of 28°C. WD of at least 1 day after inoculation (DAI) was found to be a necessity for P. fuligena infection to take place. In all the tested parameters, young and fully expanded leaves of 3 and 5 weeks age as well as the youngest leaves (1 week old) were more susceptible than leaves of 7 weeks age. Eventually, all these determinant factors, i.e. T, WD and A, influenced the incubation (IP) and latent (LP) periods. For instance, under CE both the respective IP and LP were 2.2 to 5 days and 3 to 9.6 days shorter at 28°C than at 24°C. Whereas a respective IP and LP of 11 to 13 and 12.5 to 18 days were recorded at 28°C from CE experiments, it was even shorter in one of the AE experiments lasting only 9.2 to 14.0 (IP) and 10.4 to 16.0 (LP) days. Under natural infection, however, IP extended from an average of 11 to 25 days. Abstract ii To test the effect of BLM vis-à-vis host growth, four sequential plantings were carried out in 2005. Weekly assessments of BLM incidence (DI) and severity (DS) fitted well to logistic functions. The sigmoidal shapes suggested that multiple cycles of infection occurred on leaves of tomato season-long. At times of heavy disease epidemics, a clear effect of reduced host growth, particularly in terms of healthy leaf area (HLA), was discernible from a comparison of treatments with (F) and without (NF) fungicide spray. A standardized disease severity (DS*) proportion of 0.3 from the two peak-epidemics plantings resulted in a 68% loss of HLA when treatments F and NF were compared. Comparison of healthy leaf area index of healthy plants (HLAIHP) with that of diseased plants in the F (HLAIF) and NF (HLAINF) treatments showed a respective HLAI loss of 11 and 50%. Two applications of the fungicide mancozeb at 4 and 6 weeks after transplanting (WAT) reduced BLM epidemics by 60 to 90%. Prevalence of such distinction in level of BLM epidemics amongst the plantings of 2005 led us to widen the area of investigation of the seasonal epidemics in relation to weather and yield. From the 24 fortnightly and monthly plantings, three BLM peak-epidemic periods were identified, i.e. August and September 2005 (1) and 2006 (2) and December to January 2005/06 (3). While maximum DS at the last assessment date of non-fungicide sprayed plants of these periods were 0.81, 0.50 and 0.56, the integral variable DS* was 0.30, 0.14 and 0.20 for the 1st, 2nd and 3rd peak-epidemic periods, respectively. Similarly, favorability indices of temperature (FIT) were 0.90, 0.61 and 0.66 and of relative humidity (FIRH) were 0.51, 0.35 and 0.44 for the peak-epidemic periods 1 to 3, respectively. Actual comparisons of F and NF treatments of these three peak-epidemic periods resulted in an average of 31.1% marketable yield loss. Marketable yield (MY) was negatively but poorly (r2 = 0.021) correlated with that of DS*. Integrating maximum plant height (MPH) in the model = ( + MPHbaMY −⋅⋅ ⋅ DSc *)1() , however, improved the fit (R2 = 0.35) with high significance all the parameters. The estimated coefficient of parameter c in the model suggested that a 1% DS* reduced marketable yield by ≈ 1.2%. Interestingly, the actual MY losses from the comparison of the F and NF treatments of the 1st and 3rd peak-epidemics were close to the prediction of this model. Besides BLM, early blight (Alternaria solani) occurred during the cool season plantings in October and November but was only dominant for the first 3 to 6 WAT and taken over by BLM thereafter. Taking into consideration the shared and overlapping niche of both diseases and that early blight has minor to negligible importance in the BioNetTM greenhouse, only the impact of BLM was analyzed in the seasonal dynamic study. In an effort to pinpoint the source of epidemics and design strategies of BLM management, the vertical distribution of BLM was studied across the canopy of the tomato cultivar FMTT260 in the BioNetTM greenhouse. After 16 weeks of a natural epidemic, DS of the lower Abstract iii canopy layer (0-50 cm) was 42% and significantly higher compared to 26% of the middle (51- 150 cm) and 5% of the upper (> 150 cm) layer. During the growing period of another natural epidemic, a similar distribution of BLM was observed, but higher DS and bigger lesions were detected on leaf positions 5 to 10 (in ascending order from bottom up) than on the first four leaves borne in the nursery. Further non-destructive samplings revealed the earliest BLM symptoms on leaf positions 5 to 8. When cohorts of 5 leaves were formed starting from the bottom, BLM incidence of leaves of the 1st cohort was only 79% while the next two cohorts reached 100%. In artificially inoculated plants, however, BLM was clearly more prevalent in the middle layer of the plant canopy as compared to the lower and the top part. Plants inoculated at 4 weeks after transplanting and monitored 10 days later, showed a DS of 43, 67 and 21% on the 1st, 2nd and 3rd cohorts, respectively. Thus, given equal chance of P. fuligena inoculum to infect all leaves of a tomato plant at one time, more BLM developed on fully expanded younger leaves than older ones. The high BLM severity of the lower canopy in natural epidemics is not related to the age of tomato leaves, but attributed to the proximity to substrate evaporation coupled with the down-hanging nature of FMTT260 leaves that created a confounding microclimate which led to higher relative humidity within the range of about 50-70 cm. As greenhouse improvements were mainly focused on reducing high temperature and excluding insects and vectors of viral diseases, the horizon of this research was extended to elucidate the impact of four cooling methods on epidemics of BLM and EB. These cooling methods included the fan and pad cooling system (FAD), natural ventilation (NV) using two mesh sizes (50 and 78, BioNetTM and Econet-TTM, respectively) and NV plus shading a nearly infrared pigment on the roof of a 78-mesh greenhouse (Econir). Severities of both diseases (DSTOT), i.e. black leaf mold (DSBLM) and early blight (DSEB), were positively related to that of increased cooling and RH. There was 50 to 94% and 54 to 61% more disease in the FAD greenhouse during the hot-wet and cool-dry seasons, respectively, as compared to the other three. Next to FAD were the Econir and BioNetTM greenhouses in which average seasonal temperature was 1 to 2ºC higher and relative humidity (RH) 10 to 20% less than in FAD.