Journal of Plant Diseases and Protection, 113 (3), S. 107–112, 2006, ISSN 1861-3829. © Eugen Ulmer KG, Stuttgart

Effect of autumn application of urea on saprotrophic fungi in off-season leaf litter of sour cherry and evaluation of fungal isolates to reduce primary inoculum of Blumeriella jaapii Der Einfluss einer herbstlichen Harnstoffbehandlung auf saprophytische Pilze in abgeworfenem Laub von Sauerkirschblättern und Beurteilung des Potenzials verschiedener Pilzisolate hinsichtlich einer Reduzierung des Primärinokulums von Blumeriella jaapii M. Bengtsson1*, H. Green1, N. Leroul1, H.L. Pedersen2 & J. Hockenhull1 1 Section for Plant Pathology, Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 2 Department of Horticulture, Danish Institute of Agricultural Sciences, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark * Corresponding author, e-mail [email protected] Received 22 December 2005; accepted 25 January 2006

Summary The effect of autumn application of urea on the activity and composition of saprotrophic fungi associated with sour cherry (Prunus cerasus) leaf litter infected with the leaf spot pathogen, Blumeriella jaapii, was studied under orchard conditions from the time of leaf fall to the release of ascospores, the following spring. The ability of treatment of infected leaves post leaf fall with urea or with inoculum of selected fungal strains to reduce primary inoculum (ascospores and winter conidia) of B. jaapii in spring was evaluated. Fungal activity was measured enzymatically by quantifying E-N-acetylglycosaminidase and fungal composition and frequency were evaluated following isolation at low temperature of mycelia embedded in leaf particles. Activity of the general fungal flora increased in both the urea and water treatments throughout the experimental period and a significantly higher level of activity was detected in urea-treated leaves up to 79 days after treatment. Most of the 2,146 fungal isolates recovered from the leaf litter during November-May could be placed in the following six groups: Phoma macrostoma (52.7%), other Phoma spp. (12.3%), Cladosporium spp. (13.8%), Alternaria and Ulocladium spp. (5.4%), Epicoccum purpurascens (2.9%) and Fusarium spp. (1.6%). These groups were isolated from the leaf litter throughout the experimental period and the effect of urea on fungal composition in the litter was very limited. Of these groups only the frequency of recovery of E. purpurascens was higher from urea than from water-treated leaves and this significant difference was maintained throughout the 115 days of the experimental period. Phoma macrostoma var. macrostoma, Cladosporium sp., Ulocladium chatarum, Epicoccum purpurascens and Fusarium lateritium var. lateritium were evaluated and compared with urea to reduce primary inoculum of B. jaapii. Urea reduced primary inoculum of B. jaapii in the spring by 77% and of the fungal strains, especially Cladosporium strain MB167 was found to be equally effective. Key words: antagonism, cherry leaf spot, fungal activity, fungal decomposition, fungal isolation, Prunus cerasus

Zusammenfassung Der Einfluss einer Harnstoffbehandlung im Herbst auf Aktivität und Artenspektrum saprophytischer Pilze auf abgeworfenem Laub der Sauerkirsche (Prunus cerasus), das mit dem Erreger der Sprühfleckenkrankheit, Blumeriella jaapii, infiziert war, wurde unter Plantagenbedingungen zwischen dem Laubfall und und dem Beginn des Ascosporenfluges im folgenden Frühjahr untersucht. Im Einzelnen wurde der Einfluss einer Laubbehandlung mit Harnstoff oder ausgewählten PilziJ.Plant Dis.Protect. 3/2006

solaten auf das Primärinokulum (Ascosporen und Winterkonidien) von B. jaapii im Frühjahr untersucht. Die biologische Aktivität der Pilze wurde über eine Messung der Enzymaktivität der ß-N-Acetylglucosaminidase bestimmt und Artenspektrum und -häufigkeit wurden nach der Isolierung von Pilzmycel aus Blattfragmenten bei niedrigen Temperaturen diagnostiziert. Die Aktivität der Pilze nahm sowohl in den Harnstoff- als auch in den Wasservarianten während des Untersuchungszeitraums zu und eine signifikant erhöhte Aktivität konnte in den mit Harnstoff behandelten Blättern bis zu 79 Tage nach Beginn der Behandlung festgestellt werden. Die meisten der 2.146 zwischen November und Mai aus dem Laub isolierten Pilze konnten den folgenden sechs Gruppen zugeordnet werden: Phoma macrostoma (52,7%), andere Phoma spp. (12,3%), Cladosporium spp. (13,8%), Alternaria und Ulocladium spp. (5,4%), Epicoccum purpurascens (2,9%) und Fusarium spp. (1,6%). Diese Gruppen wurden während des Untersuchungszeitraums durchgängig aus dem abgeworfenen Laub isoliert und der Einfluss der Harnstoffbehandlung auf das Artenspektrum war äußerst gering. Als einzige Art wurde E. purpurascens häufiger aus Harnstoff- als aus Wasser-behandeltem Laub isoliert und der Unterschied war während des gesamten 115tägigen Versuchszeitraums signifikant. Phoma macrostoma var. macrostoma, Cladosporium sp., Ulocladium chatarum, Epicoccum purpurascens und Fusarium lateritium var. lateritium wurden zusammen mit der Harnstoffvariante hinsichtlich ihres Potenzials zur Reduzierung des Primärinokulums von B. jaapii getestet. Harnstoff reduzierte das Primärinokulum im Frühjahr um 77% und von den untersuchten Pilzstämmen zeigte Cladosporium strain MB167 einen vergleichbaren Effekt. Stichwörter: Antagonismus, Isolierung, pilzliche Aktivität, pilzliche Diversität, Prunus cerasus, Sprühfleckenkrankheit

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Introduction

Cherry leaf spot caused by Blumeriella jaapii (Rehm) v. Arx is an important disease of sour cherry (Prunus cerasus L.) and other Prunus spp. (JONES 1995). The fungus survives the winter in off-season leaf litter, producing primary inoculum in fruiting bodies the following spring (JACOBSEN and JØRGENSEN 1986). Treatment of infected leaves in autumn with urea has previously been shown to reduce the production of primary inoculum of the fungus the following spring and to have a possible influence on fungi associated with treated leaf litter (PEDERSEN and HOCKENHULL 1996). The objective of the present study was to clarify whether urea treatment post leaf fall of naturally infected sour cherry leaves also has an effect, quantitatively and qualitatively, on the fungal community associated with decomposition of sour cherry leaves and to evaluate

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the potential of selected saprotophic fungi to reduce the amount of primary inoculum of B. jaapii. The approaches were i) to assess the activity of the general fungal community associated with sour cherry leaf litter by measuring the activity of the fungus-specific enzyme E-N-acetylglucosaminidase (NAGase) (MILLER et al. 1998), ii) study the composition of saprotrophic fungi by using a particle filtration isolation technique at low temperature for the recovery of fungal decomposers of sour cherry leaves under Danish winter conditions, and iii) to compare the inhibitory effect of urea and selected fungal isolates on the production of primary inoculum by B. jaapii in off-season leaf litter.

2 Materials and methods 2.1 Leaf material Leaves infected with B.jaapii were picked from sour cherry trees (Prunus cerasus cv. ‘Stevnsbaer’) in an orchard at the Danish Institute of Agricultural Sciences (DIAS), Aarslev, Denmark, at the time of natural leaf fall in September and October. Experiments were carried out over three years. No fungicides had been used in this orchard in recent years. The leaves were dried between filter paper for two days at room temperature and stored in plastic-coated paper bags at 4°C until used.

2.2 Investigations on fungal activity and frequencies in leaves 2.2.1 Treatment and sampling of leaves. The dried leaves were soaked in water for 15 minutes before being placed with the adaxial leaf surface up on nylon mesh (mesh size 0.65 mm-2) mounted on a wooden frame (50 x 50 cm) then held in place with chicken netting. The leaves were sprayed with a hand sprayer on both leaf surfaces until run-off with water (control treatment) or water containing 2.5% urea (w/v, Sigma Chemical Company, St. Louis, MO, USA). The leaves were incubated at 5-8°C for 3 days. At the end of November the wooden frames were placed in a randomised block design on the ground under sour cherry trees at DIAS. Each frame contained 125 leaves, and each treatment had eight frames arranged in four blocks. The leaves were incubated in the orchard until the time of bloom of the cherry trees, the following spring. Eighteen leaves were sampled randomly within the two replicate frames from each treatment and each block on the day of urea application and thereafter on day 3, 7, 16, 58, 79, 115 and 163 (beginning of bloom of sour cherry). The leaves were transported to the laboratory in plastic bags in a cooler bag (4°C; for maximum 3 hours). On arrival at the laboratory samples were unpacked, placed on filter paper and dried in a laminar airflow bench over night at room temperature after which they were stored in sealed plastic bags at 4°C until processing.

2.2.2 NAGase assay. Enzyme activity measurements were carried out on day 0, 3, 7, 16, 58, 79 and 115. Dried leaf samples were placed in a sterile blender (Waring model 32BL80) and pulverised at high speed for 15 seconds. Of the homogenised leaf samples 0.16 g was placed into a 8 ml centrifuge tube. The assay was conducted for 60 min at 25°C in 2.5 ml maleate-NaOH buffer (50 mM, pH 6.8) containing 0.2 mM of 4-methylumbelliferyl-N-E-D-glucosaminide (CN Biosciences, Inc., San Diego, CA, USA). The assay was quenched by addition of 2.5 ml ice-cold 96% ethanol. Leaf particles were pelleted at 13,000 x g for 12 min at 4°C, and 2.5 ml of the supernatant was transferred to 500 Pl tris buffer (2.5 M, pH 10). The mixture was filtered through a 0.45 Pm Minisart cellulose acetate filter (Sartorius, Goettingen, Germany) into plastic cuvettes (Kartell, KEBO Lab., Albertslund, Denmark). The flu-

orescence emitted by the enzymatically released 4-methylumbelliferone (MU) moiety was monitored with a luminescence spectrometer (LS50B; Perkin-Elmer Ltd., Buckinghamshire, United Kingdom) by excitation at 365 nm and reading at 448 nm. The readings were corrected for quenching (FREEMAN et al. 1995) and non-enzymatic hydrolysis of 4-methylumbelliferyl-N-E-D-glucosaminide and converted to nanomoles of MU per minute per gram of dry leaf. Triplicate measurements per replicate were performed.

2.2.3 Particle filtration isolation. Isolation of fungal populations was done using SNA (18.4 g/l special low nutrient agar, ADSA Micro, Barcelona, Spain, amended with 5 g/l bacteriological agar (Difco Laboratories, Detroit, MI, USA) and 250 mg/l Novobiocin). Identifications were made from colonies sub-cultured on PDA (potato dextrose agar, Difco) amended with 5 g/l bacteriological agar (Difco), in small Petri plates (Ø = 55 mm). Particle filtration isolation was carried out on day 3, 16, 58, 79, 115 and 163. The isolation procedure was modified from the method described by BILLS and POLISHOOK (1994). Dried leaf samples were placed in a sterile blender (Waring model 32BL80) and pulverised at high speed for 15 seconds. Replicates were pooled and mixed, and a sample (0.85 g) was washed with water through 1 mm and 0.2 mm sieves and then through a sterile polyester 0.1 mm mesh filter (PET 100 HD, Streno, Farum, Denmark). Each wash was carried out for 10 min. The particles trapped on the 0.1 mm polyester filter were gently transferred to a sterile vial and suspended in sterile water. Particles were re-suspended by gentle agitation and aliquots were distributed to eight 50 ml conical centrifuge tubes with screw caps filled with sterile water. Between 0.5–0.75 ml settled particles per tube was found to be a suitable density. While the amount of particles varied slightly from sample date to sample date, the same amounts settled in all eight tubes in samples from the same sample date. Settled particles were washed by decanting the water and adding fresh sterile water giving a final volume of 50 ml. Washing was repeated twice. From each vial of suspended particles dilutions in water of 1:10 and 1:100 were prepared. From each leaf sample 0.1 ml aliquots of particle suspension were pipetted into Petri plates and approximately 12 ml of molten (45°C) SNA was poured into each plate. Plates were gently swirled to distribute the particles evenly in the agar. Two plates were prepared from the initial suspension and two from each of the dilutions. The plates were incubated at 5°C in darkness. After 4 days of incubation the first hyphal growth appeared. For each treatment/sampling time plates were selected and isolation of all hyphal tips emerging from all the particles in the plate was attempted. Every second day for the next two weeks the plates were inspected using a dissecting microscope and each particle with emerging hyphae was cut from the agar with a needle and transferred for sub-culturing and later identification. A maximum of 25 such particles from each of the two replicates per leaf sample were transferred. A total of 400 particles from each date of isolation were transferred for sub-culturing. The transferred particles were incubated for one week at 10°C in darkness and then checked for purity of the emerging fungus. In most cases only one fungal species developed but an additional sub-culturing was carried out when more than one fungal species grew out of a particle. Subcultures were incubated for 6 weeks at 10°C and then at room temperature under near-UV light for an additional two weeks. On the basis of sporulation or differential growth patterns isolates were sorted into species, morphologically similar groups or into a group of non-sporulating isolates. Isolates that were difficult to group were re-cultured on SNA. For each date of isolation and treatment the relative frequency of predominant groups of fungal strains was calculated using the formula: percentage frequency = number of isolates in a group x 100/total number of isolates recovered on that sampling date. J.Plant Dis.Protect. 3/2006

NAGase activity (nmol MU/min/g leaf)

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(1998) except that one drop Tween-80 (Sigma) was added to the water (35 ml), and that following filtration, leaf particles were washed with an additional 10 ml water. Spore quantification was based on three samples for each net bag. The spore production of each treatment is presented as the number of ascospores and winter conidia per gram leaf material.

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Fig. 1: The fungal E-N-acetylglucosaminidase (NAGase) activity in leaf litter of sour cherry treated with water or urea post leaf fall. A fluorogenic assay was used to quantify the release of 4-methylumbelliferone (MU) from the 4-methylumbelliferyl-N-acetyl-E-D-glucosaminide substrate at seven sampling dates during winter (November – March). Bars represent the standard error.

All data were analysed statistically using SAS (Statistical Analysis System, version 6.12, SAS Institute, Cary, NC, USA). Data from the NAGase assay were transformed logarithmically to obtain variance homogeneity. Levels of significance for the main treatments and their interactions were calculated using the General Linear Models Procedure (PROC GLM). Isolation data were analysed for variance between treatments for each fungal group and sampling date using the GLM Procedure. For data on primary inoculum from the preliminary screening experiment standard deviations were calculated. Data on primary inoculum from the last year were transformed logarithmically to obtain variance homogeneity and analysis of variance was performed to test the effect of fungal isolates and urea on primary inoculum production using the GLM procedure. Least significant differences (LSD) were used to detect significant differences between means.

2.3 Evaluation of fungal isolates to reduce primary inoculum of Blumeriella jaapii 3 A preliminary screening of candidate fungal strains was carried out. Twenty-one fungal isolates belonging to the taxa Alternaria spp., Cladosporium spp., Epicoccum purpurascens, Fusarium spp., Phoma macrostoma, Phoma spp. and Ulocladium spp. isolated during the first year of the study were screened for their ability to reduce the amount of primary inoculum produced by B. jaapii in leaf depots. The isolates were grown at 20°C on potato dextrose agar (PDA, Difco) in Petri plates for 3 weeks. Inoculum of each isolate was harvested by scraping off mycelium and spores from one to two replicate plates for each isolate. The suspensions were homogenised in an Ultra-Turrax T25 (IKA-Labortechnik, Staufen, Germany) for 1 min at 11,000 rpm and the volume of each isolate was adjusted to 40 ml with sterile water. The dried leaves were soaked in tap water for 15 minutes, 50 leaves were placed in plastic net bags (27 x 35 cm; 1 cm2) and each net bag received the inoculum from one isolate. Water and 5% (w/v) urea were used as reference treatments. The net bags were placed in wooden frames on Vatex matting (Tambour Tex, Randers, Denmark) and incubated under a plastic cover at 15°C (12 hours light/12 hours darkness) for 5 days and then placed randomly on a gravel area at DIAS until the following spring. On the basis of the preliminary screening the five most promising antagonistic strains were selected for further testing and also for conformation of their identification at the Centralbureau voor Schimmelcultures, CBS, Baarn, the Netherlands. These strains were Phoma macrostoma var. macrostoma (MB146), Epicoccum purpurascens (MB232), Fusarium lateritium var. lateritium (MB256), Cladosporium sp. (MB167) and Ulocladium chartarum (MB50). Inoculum was produced as above, except that inoculum was harvested from eight replicate plates for each strain, the suspensions were homogenised in a Waring Blender for 1 min at high speed and the volume of each strain was adjusted to 500 ml with sterile water. Water and a 2.5%(w/v) urea solution were used as reference treatments. The leaves were treated and incubated as described above except that incubation at DIAS took place on soil in a cherry orchard in a randomised block design with four replications until the following spring. In both experiments treated, over-wintered leaves were returned from the orchard in the beginning of May, dried overnight and stored in plastic bags until processed. After the petioles were discarded the dry weight of the total content of each net bag was determined. Ascospore and winter conidia production were estimated using the water bath method of Kollar J.Plant Dis.Protect. 3/2006

Results

3.1 Fungal activity Fungal activity, measured with the NAGase reaction, increased significantly (P < 0.001) in both the water- and urea-treated leaves throughout the experimental period (i.e. 115 days, Fig. 1). For the water-treated control the increase of the NAGase activity was linear (r2 = 0.73; P < 0.001) starting at 0.7 nmol MU min-1 g-1 at day 0 and ending at 16.2 nmol MU min-1 g-1 at day 114. For the urea treatment, a similar linearity (r2 = 0.71; P < 0.001) was observed until day 79. In this case the increase in the NAGase activity was from 3.0 nmol MU min-1 g-1 at day 0 to 16.5 nmol MU min-1 g-1 at day 79, where the activity stabilised at a constant level. NAGase activity in the urea-treated leaves was significantly (P = 0.003) higher than on the water treated leaves at day 3, 16, 58 and 79. Sampling dates and treatments interacted significantly (P < 0.001).

3.2 Fungal diversity Of the 2400 transferred particles a total of 2146 culturable fungal isolates were recovered from six dates of collection during the winter from leaf litter of both treatments. Coelomycetes dominated and accounted for at least 66.5% of all isolates, followed by Hyphomycetes (26.3%). The most dominant species was Phoma macrostoma, which accounted for 52.7% of all isolations (Fig. 2a). Cladosporium spp., 13.8% (Fig. 2b) and Phoma spp. (excluding Phoma macrostoma), 12.3% (Fig. 2c), were the next most abundant groups while groups containing species of Alternaria and Ulocladium accounted for 5.4% (Fig. 2d), Epicoccum purpurascens for 2.9% (Fig. 2e), followed by Fusarium spp. with 1.6% (Fig. 2f). Among the fusaria, F. lateritium dominated. Of the remaining isolates Acremonium spp., Ascochyta spp., Aurobasidium pullulans, Penicillium spp. and yeast-like isolates were most abundant and accounted for about 1% each. The remaining 5.7% of the total isolates consisted of unidentified species.

3.3 Fungal frequencies The frequencies of P. macrostoma were consistently high and no significant differences were found between treatments,

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Fig. 2: The frequencies of six predominant fungal groups isolated as mycelial fragments from sour cherry leaf litter, treated with water or urea post leaf fall, recovered from six sampling dates during winter (November – May): A. Phoma macrostoma, B. Cladosporium spp, C. Phoma spp. (excluding Phoma macrostoma), D. Alternaria and Ulocladium spp. E. Epicoccum pupurascens, F. Fusarium spp. Bars represent the standard error. except for the last day of sampling (day 163), when the frequency in the urea treated leaves was significantly lower (P = 0.012) (Fig. 2a). The frequency of Phoma spp. (excluding Phoma macrostoma) (Fig. 2c) on day 3 was significantly lower for the urea treatment (P = 0.037) but no differences were found between treatments for the other days. For both treatments a significant increase in the frequency of Phoma spp. was found on day 163 (P < 0.001), where the mean frequency was 22.8%. The frequency of Cladosporium spp. (Fig. 2b) increased significantly (P < 0.001) from day 3 to day 16 for both the control and the urea treatment to 46.9% and 32.8%, respectively, and subsequently the frequencies decreased ending at 2.8% and 1.5%, respectively, on day 163. The frequency of Alternaria and Ulocladium spp. (Fig. 2d) increased significantly from 0% to 8.2% on day 58 for both treatments and decreased slightly to 6% at day 163, with no significant differences between treatments. For E. purpurascens (Fig. 2e) the frequency for both treatments increased from 0% at day 3 to 2-5% for the remaining period and significant higher frequencies were observed for day 58, 79 and 115 (P = 0.003, P = 0.034 and P = 0.010, respectively) from urea-treated leaves, while no significant difference was observed at day 163. The fusaria (Fig. 2f) increased from 0% (day 16) to 2.2% (day 115) with no significant difference between the treatments. At day 163 the frequency of fusaria in the urea-treated leaves

declined to 0%, while the frequency for the control remained at 2%. The frequencies of all other fungal species were about 5% all through the sampling period except for day 3 and day 163. On day 3 the frequency was more than 20%, due to the presence of mainly Aureobasidium pullulans and Ascochyta spp. in both treatments, and on day 163 the frequency of other fungal species increased due to the abundant occurrence of Acremonium spp. on this day (data not shown). No difference was observed in the occurrence of other minor species between the control and the urea treatments (data not shown).

3.4 Evaluation of fungal isolates to reduce primary inoculum of Blumeriella jaapii Twenty-one fungal isolates belonging to the predominant fungal groups found in the present study were initially screened and compared with urea for ability to reduce production of primary inoculum by B. jaapii (Fig. 3). The 5% urea treatment reduced the spore production by more than 90%. While not all strains within the groups were equally effective, each group contained one or more strains that reduced spore production by more than 50%, i.e. strain MB146 (Phoma macrostoma var. macrostoma), MB167 (Cladosporium sp.), MB50 (Ulocladium chartarum), MB174 Alternaria/Ulocladium sp.), MB184 (AlJ.Plant Dis.Protect. 3/2006

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Fig. 4: Effect of water (control), urea (2.5%) and five fungal isolates Cladosporium sp. (MB167), Fusarium lateritium var. lateritium (MB256), Ulocladium chartarum (MB50), Epicoccum purpurascens (MB232) and Phoma macrostoma var. macrostoma (MB146) on the production of primary inoculum (ascospores and winter conidia) of Blumeriella jaapii assessed after over-wintering. Treatments were applied post leaf fall as spore-mycelia suspensions. Coloumns with the same letters are not significantly different (P < 0.05). ternaria/Ulocladium sp.), MB232 (Epicoccum purpurascens), MB76 (Fusarium lateritium) and MB256 (Fusarium lateritium var. lateritium). In the following year when the selected strains were retested and compared with 2.5% urea significant spore reductions were obtained for the urea treatment and with MB167 (Cladosporium sp.), MB50 (Ulocladium chartarum) and MB256 (Fusarium lateritium var. lateritium) (Fig. 4). Leaves treated with urea produced 77.2% fewer spores than the water-treated control. The strain of Cladosporium sp. (MB167) significantly reduced spore production by 75.7% (i.e. to the same level as the urea treatment). The treatments with the strains of Fusarium lateritium var. lateritium (MB256) and Ulocladium chartarum (MB50) significantly reduced spore production by 43.5% and 34%, respectively. No significant reduction in spore amount was recorded with strains MB232 (Epicoccum purpurascens) and MB146 (Phoma macrostoma var. macrostoma) (Fig. 4). J.Plant Dis.Protect. 3/2006

Application of urea to off-season leaf litter is widely understood to enhance the decomposition process and its use is recommended for reduction of primary inoculum of the scab fungus Venturia inaequalis in apple (MACHARDY 1996). Earlier studies on the modes of action of urea against the over-wintering stage of V. inaequalis indicated a fungistatic effect on pseudothecia formation by the pathogen in senescent apple leaves and an indirect effect due to a stimulation of fungal and bacterial populations associated with the apple leaf litter (ROSS and BURCHILL 1968; CROSSE et al. 1969; BURCHILL and COOK 1971). Urea has been shown to reduce the development and maturation of primary fruiting bodies of B. jaapii in sour cherry leaves but also to stimulate some fungal genera in urea-treated leaves (PEDERSEN and HOCKENHULL 1996). In the present study, leaf pulverisation followed by leaf particle-filtration (BILLS and POLISHOOK 1994) and low temperature incubation of leaf particles embedded in agar (CARREIRO and KOSKE 1992; BERNIER et al. 1996) were used in order to increase the likelihood of isolating fungi involved in decomposition of sour cherry leaf litter under orchard conditions. The composition of the recovered taxa, however, was similar to that reported by workers using procedures such as dilution plating of homogenates of leaf litter or leaf surface washings (ANDREWS and KENERLEY 1979; HERING 1965; KUTER 1986; BERNIER et al. 1996) or direct observation and transfer of propagules with dissecting tools (HOGG and HUDSON 1966; ANDREWS and KENERLEY 1979; BERNIER et al. 1996). It is thus interesting, that even though the fungi recovered in the present study were isolated as mycelial fragments, abundantly sporulating taxa including Alternaria, Cladosporium, Epicoccum, Fusarium, Phoma and Ulocladium were the most abundant groups. Our findings are thus in close accord with other reports on the composition of fungal communities involved in decomposition of leaf litter (HERING 1965; HOGG and HUDSON 1966; ANDREWS and KENERLEY 1979; KUTER 1986; BILLS and POLISHOOK 1994; BERNIER et al. 1996). Quantification of the NAGase activity (RAST et al. 1991; CIRANO and PEBERDY 1993) was used in the present study to measure the general activity of the fungal community in cherry leaf litter following treatment with water or urea. Immediately after treatment fungal activity was significantly higher in the water-treated control than in the urea-treated leaves. From then onwards the situation was reversed with a significant increase in activity in the urea-treated leaves from day 16 until day 79. The differences in activity at the start of the treatments could be that the fungal community in the

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leaves grows more rapidly in the water-treated leaves than in the urea-treated ones because of the inhibitory effects of urea on the saprotrophic fungi. As urea is reduced to ammonia and water, this inhibitory effect would be replaced by stimulation due to the metabolization of the released N by surviving leaf fungi. The finding in the present study that the compositions of the fungal community in the urea- and water-treated leaves were very similar suggests that fungal inhibition by urea in the cherry leaves is fungistatic rather than fungitoxic. These findings are supported by results of a study on the influence of urea on bacterial populations associated with decomposing cherry leaves infected with B. jaapi (H. GREEN et al., in preparation). Even though B. jaapii grows saprotrophically for a period shortly after leaf fall before formation of its over-wintering structures (BENGTSSON 2001) it was not isolated from water-treated leaves. However, although B. jaapii can be cultured on synthetic media it grows very slowly (BENGTSSON 2001), which may explain why it was not recovered in this study. Phoma macrostoma dominated at all isolation dates by its frequency of more than 40% and Cladosporium spp. had a high frequency in both urea- and water-treated leaves, but only at the beginning of the experiment. In the studies by PEDERSEN and HOCKENHULL (1996) in cherry and BURCHILL and COOK (1971) in apple, the number of colonies of Cladosporium spp. increased over time and was higher for urea-treated leaves. Both of these studies were carried out using dilution plating of leaf washings, which might have favoured the higher Cladosporium counts. Among the most predominant fungal groups found in the present study, E. purpurascens was stimulated by urea. No clear effect was observed for any of the other groups of fungi involved in decomposition of sour cherry leaf litter. The isolates selected to be evaluated as potential biocontrol agents all belonged to the main groups of saprotrophic species isolated from the cherry leaf litter. Only the isolate of Cladosporium sp. (MB167) was able to reduce primary inoculum of B. jaapii considerably and to the same level as the urea treatment. In addition, it was observed in spring that leaves treated with Cladosporium sp. or urea appeared more decomposed than any of the other treatments (data not presented). Beside its ability to decompose leaves this strain may also have the ability to colonise leaves before leaf fall, since species of this genus are frequently isolated from the phylloplane and considered as primary saprotrophs (HUDSON 1971; DICKINSON 1975). In this study treatment of sour cherry leaves post leaf fall with urea was shown to have influenced the fungal community quantitatively as indicated by the increased activity of the general fungal flora. However, it seemed to have had little influence on the diversity of the fungal community, shown by the largely unaltered composition of predominant fungal groups isolated from sour cherry leaf litter treated with urea. One strain of Cladosporium sp. was identified as a potential agent for biological control of cherry leaf spot and further studies are needed to elucidate its practical potential.

Acknowledgements This study was financed by a grant from the Danish Ministry of Foods, Agriculture and Fisheries as a part of the programme: ‘Biological and Microbiological Control of Pests’. The technical assistance from Anita Idoff is highly appreciated.

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