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Effect of environmental factors on growth, pycnidial production and spore germination of Microsphaeropsis isolates with biocontrol potential against apple scab
Mycol. Res. 106 (12): 1455–1462 (December 2002). f The British Mycological Society
1455
DOI: 10.1017/S0953756202006858 Printed in the United Kingdom.
Effect of environmental factors on growth, pycnidial production and spore germination of Microsphaeropsis isolates with biocontrol potential against apple scab
Odile CARISSE* and Julie BERNIER Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, St-Jean-sur-Richelieu, Quebec J3B 3E6, Canada. E-mail : [email protected] Received 21 Febuary 2002; accepted 30 September 2002.
The potential as biocontrol agents of the apple scab pathogen, Venturia inaequalis and the effect of environmental factors on growth, pycnidial production and spore germination of four isolates of Microsphaeropsis were examined on culture media and leaf discs. V. inaequalis ascospore production was reduced by 52, 77, 89 and 95 % on leaf discs treated with the isolates IMI 294735, P176A, DAOM 198536 and P130A, respectively. For all isolates, optimum temperature for mycelial growth was 25 xC. More pycnidia were produced on culture media than on leaf discs. On culture media, optimum temperature was from 15 to 25 x, while on leaf discs it varied among isolates. Pycnidial production was inhibited by darkness and a minimum of 8 h light dx1 was required. Spores of isolates P130A and DAOM 198536 germinated at temperatures above 15 x and at pH 4–5, as compared to 10 x and pH 4–8 for isolates P176A and IMI 294735. The best candidates were P130A and DAOM 198536 and optimum conditions for growth, pycnidial production and spore germination were temperatures between 20 and 25 x, pH approx. 4 and light.
INTRODUCTION Apple scab, caused by Venturia inaequalis, is one of the most important diseases of apple and the most costly to control (Sivanesan & Waller 1974, MacHardy, Gadoury & Gessler 2001). Since the development of organic fungicides in the 1940s, fungicides became the sole means for managing this disease. Over the years, there have been several attempts to develop biocontrol strategies, but until recently, none had reached the point where it could be used commercially (Carisse & Dewbney 2002). V. inaequalis overwinters as incipient ascomata formed in apple leaf litter. In the spring, when leaf litter is wetted by rain, mature ascospores are forcibly discharged into the air. The ascospores are dispersed by wind from the ground to the newly emerged leaves. There, the ascospores germinate, penetrate and form lesions. In the autumn, V. inaequalis becomes a saprophyte and overwinters as ascomata initials. The most promising approach to apple scab biocontrol consists in applying a biocontrol agent in the autumn, that will interrupt overwintering of the teleomorph (MacHardy et al. 2001, Carisse & Dewdney 2002). Bernier, Carisse & Paulitz (1996) isolated 189 fungi from dead apple leaves and Philion, Carisse & Paulitz * Corresponding author.
(1997) screened some of these for their ability to inhibit ascospores production in in vitro tests. From this evaluation, five isolates, including two Microsphaeropsis (P130A and P176A), were selected. These potential biocontrol agents were further tested under orchard conditions with the most reliable reduction in ascospore production being obtained with isolate P130A (Carisse et al. 2000), described as the new species M. ochracea (Carisse & Bernier 2002). Based on a study of the interaction between M. ochracea and V. inaequalis (Benyagoub, Benhamou & Carisse 1998), it was assumed that M. ochracea must penetrate the leaves in order to interact with the formation of ascomata by V. inaequalis. However, information on the ecology of Microsphaeropsis species is sparse, and non-existent for M. ochracea. It is not clear whether M. ochracea or other Microsphaeropsis species colonize and penetrate the apple leaves and produce pycnidia when applied in the autumn on senescent apple leaves, either on the tree canopy (after harvest but before leaf fall) or on the ground. The potential of Microsphaeropsis isolates to reduce ascospore production by V. inaequalis has been demonstrated under controlled and small plot conditions (Carisse et al. 2000). For commercial uses, proper timing of the antagonist is crucial. The autumn treatment should be made so that all leaves are treated and to
Microsphaeropsis : biocontrol and environmental factors favour colonization by the antagonist in order to maximize the possibility of contact between the antagonist and V. inaequalis. If the antagonist is applied on the tree canopy before leaf fall (soon after harvest) and if commercial sprayers are used, most leaves should be sprayed but the environment at the leaf level may not favour leaf colonization by the antagonist or allow it to maintain its population at a high level. On the other hand, if the antagonist is applied on the ground after leaf fall, all leaves may not be reached and weather conditions in northern regions may be unfavourable to antagonistic activity. Before deciding which spray strategy is most promising, it is important to determine the effect of the environment on fungal growth, pycnidial production, and spore germination. The objective of this study was to compare the potential of four Microsphaeropsis spp. as biocontrol agents of apple scab and to obtain quantitative information on the effect of temperature, pH and light on fungal growth, pycnidial production and spore germination of M. ochracea and three related species.
MATERIALS AND METHODS Source of isolates and inoculum production Four isolates, designated as M. ochracea P130A, Microsphaeropsis sp. P176A, Microsphaeropsis sp. DAOM 198536 and M. arundinis IMI 294735 were included in this study. The first two isolates were isolated from dead apple leaves collected in Quebec orchards (Bernier et al. 1996) and stored in water or soil at 4 xC ; P130A is deposited at ATCC 74412. The other two isolates also originated from apple leaves or fruits and were obtained from the Canadian Fungal Culture Collection (DAOM, Ottawa) and the CABI Bioscience UK Centre (IMI, Egham). John Andrews (Wisconsin University) kindly provided the culture of Athelia bombacinai, which was used as a positive control (Heye 1982). Effect of isolates on ascospore productions Disease-free apple leaves were collected from Malus pumila cv. ‘ McIntosh’ trees. Leaf discs 2.6 cm diam were cut and sterilized by irradiation (10 h under 40 kGy [4 Mrad] gamma radiation). Petri dishes 9 cm diam were filled with 20 ml of autoclaved PerliteTM (Perlite Atlas, Beauport, QC) and 10 ml of sterilized distilled water. The irradiated leaf discs were placed in the Petri dish on perlite (2 discs per dish). A mycelial suspension of V. inaequalis was made from a mixture of ten isolates, all originating from scabbed leaves. A mixture of isolates was used to increase the number of pairs of compatible mating types, and consequently ensure the production of ascomata. Each isolate was previously grown on PDA at 18 x. As the growth rate of each isolate was quite different, when the isolates covered more than twothirds of the surface of a 9 cm Petri dish they were stored at 2 x until inoculation of the leaf discs (max. 2 wk). To
1456 prepare the mixture of V. inaequalis isolates, the content of one Petri dish of each isolate was placed in a sterile plastic bag (Steward Medical, London) and 200 ml of sterile water was added. The content of the bag was homogenized in a Stomacher TM laboratory blender (Seward Medical) for 6 min. Each leaf disc was inoculated with 150 ml of the V. inaequalis mycelial slurry. Immediately after the disc inoculation with V. inaequalis, the discs were inoculated with the four isolates of Microsphaeropsis sp. at a rate of 100 ml of a spore suspension of 4.5r108 spores mlx1. Athelia bombacina (positive control) was stored on PDA slants under mineral oil maintained at 2 x. Prior to the disc inoculation, the fungus was cultured on PDA and mycelial slurry was made from 3-wk-old cultures as described for V. inaequalis. Each disc was inoculated with A. bombacina at a rate of 100 ml of mycelial slurry per leaf disc. For each isolate, eight Petri dishes were inoculated and the control consisted of eight dishes of leaf discs inoculated only with V. inaequalis. The experiment was set up as a completely randomized design with eight repetitions and the entire experiment was conducted twice. Several supplementary controls were prepared and used to follow the maturation of ascomata and to determine the optimal time for ascospore extraction. The leaf discs were incubated at room temperature for 3 wk, then transferred to a cold room adjusted to 4 x with 90% relative humidity in complete darkness for 4 wk, followed by 12 wk at 10 x, with 90 % relative humidity and complete darkness. When ascomata were mature in the controls, all dishes were brought to the laboratory and kept at room temperature (20–25 x) for another 3 wk to favour ascospore maturation. To evaluate ascospore production per leaf disc, two discs were placed in a 50 ml Falcon test tube filled with 20 ml of distilled water. A bubbler type apparatus, developed by Philion et al. (1997) was used to force ascospore ejection into the water. Air was forced at the rate of 25.5 l minx1 into the Falcon tubes through Pasteur pipettes for a total of 6 h. The aliquot of each tube was decanted into another tube and a few drops of Lugol’s iodine solution were added to each ascospore suspension to avoid germination. The contents of the tubes were centrifuged at 3800 rev minx1 for 10 min (Accuspin FR, Beckman, CA). The liquid was carefully drawn out of the tubes and 5 ml was conserved so that the ascospore pellet and surrounding liquid were not disturbed. Ascospore concentration was estimated using a haemocytometer and ascospore productivity reported as ascospores cmx2 of leaf disc. Effect of temperature on mycelial growth on culture media The inoculum of Microsphaeropsis isolates was produced by growing them on potato dextrose agar (PDA ; Difco, MD) at room temperature for 2 wk. The spore suspensions were made by scraping and transferring pycnidia into a 0.85 % saline solution. The suspensions
O. Carisse and J. Bernier were adjusted to 1r106 spores mlx1 using a haemocytometer. The effect of temperature on mycelial growth was evaluated on PDA culture media. A mycelial plug 6 mm diam was removed from the periphery of an actively growing culture and placed in the centre of a PDA dish. Dishes were incubated at different temperatures and radial growth was measured after 1 and 2 wk of incubation. For each fungus, four dishes were inoculated and the experiment was conducted twice. Effect of temperature, pH and light on pycnidial production and of temperature and pH on spore germination on culture media To study pycnidial production on culture media, 50 ml of spore suspension was inoculated on PDA Petri dishes and spread onto the entire surface of each dish using a glass rod. For each treatment, four Petri dishes were inoculated. To test the effect of temperature, dishes were incubated for 3 d at room temperature (20–25 x) to allow hyphal growth prior to transfer to controlled-environment growth chambers (Percival, Bonne IW ; model I-30BLLX) adjusted to temperatures of 5, 10, 15, 20 and 25 x (¡0.5 x) with a photoperiod of 12 h light dx1 provided by incandescent and fluorescent lights (Vita Lite, Duro-Test, Montreal). The light was adjusted to 1950–80 lx in all incubators using a LI-1000 data logger (LI-COR, NE). To test the effect of pH, PDA medium was buffered prior to autoclaving with a citrate-phosphate solution to obtain pH values of 4, 5, 6 and 7, and with a phosphate buffer solution to obtain a pH of 8. When adjusting the medium to pH 4, care was taken to avoid acid hydrolysis, which prevents gelation of agar following cooling. The citrate-phosphate buffer solution and the PDA medium were autoclaved separately and then aseptically mixed together during cooling. The buffer solutions were prepared following the method in Dhingra & Sinclair (1985). The pH was measured at the time of inoculation of dishes and at the end of the experiment to make sure that it had been maintained throughout the experiment. The inoculated dishes were incubated in controlled-environment growth chambers adjusted to 20 x with a photoperiod of 12 h light as described above. To test the effect of light regime, the inoculated dishes were incubated at 20 x under daylight of 0, 8, 12, 16 and 24 h provided by fluorescent and incandescent lights (as described above). The same protocol as described above was used to study the effect of temperature and light regime on spore germination on culture media with the following modifications: the spore suspensions were spread onto water agar (WA ; Difco, MD), 15 g lx1, instead of PDA. Secondly, the dishes were transferred to the growth chambers adjusted to different temperatures immediately after inoculation and five replicates were used per treatment. To evaluate pycnidial production, the inoculated dishes were removed from the incubators and plugs
1457 3 mm diam ; two plugs per plate, were cut at random in the fungal growth area and removed from the mycelial mat. The plugs were transferred to 500 ml of sterile distilled water in 2 ml Eppendorf tubes placed in boiling water for 15 min to melt the agar. The mycelial mat containing the pycnidia was then transferred on a glass slide and the total number pycnidia per plug counted with a microscope at r25. Samples were collected every 2–3 d over a period of 14–18 d, depending on experiments. Spore germination was assessed after 16, 24 and 40 h of incubation using an inverse microscope adjusted to r250. A total of 100 spores from each dish were examined and a conidium was considered germinated when the germ tube was at least the length of the spore. Effect of temperature and light on pycnidial production on apple leaf discs The effects of temperature and light on pycnidial production were examined on apple leaf discs. Leaf discs 2.6 cm diam were cut from green apple leaves, cv. McIntosh, using a cork borer. The discs were sterilized by irradiation (10 h under 40 kGy [4 Mrad] gamma radiation). Irradiated leaf discs were stored at 4 x in hermetically closed glass jars until needed for the experiments. To provide a moist environment but to avoid the contact between the fungi and the media, the leaf discs were placed on previously autoclaved wood coffee sticks placed on the surface of WA dishes with two discs per dish. For the four isolates, spore suspensions were prepared as previously described and 100 ml of suspension was inoculated on each leaf disc. To test the effect of temperature, the dishes were incubated for 3 d at room temperature to allow hyphal growth prior to transfer to the controlled-environment growth chambers adjusted to different temperatures with light adjusted to 1720–1885 lx. To test the effect of light regime, the inoculated dishes were incubated at 20 x under daylight of 0, 8, 12, 16 and 24 h provided by fluorescent and incandescent light. The density of pycnidia on each leaf disc was evaluated by counting the number of pycnidia in two binocular fields, which corresponded to an area of 1.36 cm2 (25.52 % of the surface of the disc). Observations were made every 2–3 d over a period of 13–15 d depending on the experiment. Statistical analysis All experiments were conducted following a completely randomized block design with 4–8 blocks (dishes), depending on the experiment, and were conducted twice. An F-test was used to determine if the variances of the two experimental runs were homogenous and if data could be pooled. Analysis of variance was used to test the effect of fungal isolates on ascospore production and of temperature, pH and light regime on mycelial growth, pycnidial production and spore germination. Significant
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Effect of isolates on ascospore production The homogeneity of variance test indicated that data from both experimental runs could be pooled (P>0.05) ; hence the analysis was conducted on pooled data. All Microsphaeropsis isolates and A. bombacina significantly reduced ascospore production (P<0.0001) (Fig. 1). An average of 1146.54 ascospores cmx2 of leaf disc was produced on the untreated discs (V. inaequalis only) and this amount was reduced by 51.99, 77.36, 88.86, 93.71 and 95.18 % on leaf discs treated with the isolates IMI 294735, P176A, DAOM 198536, A. bombacina and P130A, respectively. There was no significant difference in ascospore production on leaf discs treated with A. bombacina and Microsphaeropsis P130A or DAOM 198536. Effect of temperature on mycelial growth on culture media Homogeneity of variance tests indicated that data from both experimental runs could be pooled (P>0.05), hence the analysis was conducted on pooled data. For all isolates, after 14 d incubation, mycelial growth increased with increasing temperature following a similar pattern (Fig. 2). Temperature had a significant effect on mycelial growth of all Microsphaeropsis isolates (P<0.0001). At 5 x, there was no significant difference between growth in mm for the four isolates (Fig. 2). At temperatures above 5 x, isolates P176A and IMI 294735 grew significantly more than the other two isolates
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(P<0.0001) and at temperatures of 15 and 25 x isolate IMI 294735 showed significantly higher growth than P176A (P<0.0001) (Fig. 2). Effect of temperature, pH and light on pycnidial production and of temperature and pH on spore germination on culture media For all tests, variances were homogenous (P>0.05), hence the analysis was conducted on pooled data. Overall, temperature had a significant effect on pycnidial production cmx2 (P<0.0001). For all isolates after 15 d of incubation, only few pycnidia were produced at 5 x and isolate IMI 294735 produced significantly more pycnidia cmx2 than the other isolates (Fig. 3). At 10 x, DAOM 198536 and IMI 294735 produced significantly more pycnidia cmx2 than the other isolates (Fig. 3). At temperatures of 15, 20 and 25 x, there was no significant
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Fig. 5. Effect of light regime on the number of pycnidia produced on PDA after 14 d incubation for four isolates of Microsphaeropsis. Bars with the same letters are not significantly different according to the least significant difference test (at the Pf0.05 level of confidence).
difference between the numbers of pycnidia produced by the four isolates (P<0.0001) (Fig. 3). Overall, pH had a significant effect on pycnidial production cmx2 (P<0.0001) but large variations were observed among observations. For all isolates after 16 d incubation, the optimum for pycnidial production was pH 4, and for most isolates only a few pycnidia were produced above pH 4. IMI 294735 differed from the other isolates producing a large number of pycnidia cmx2 at pH 5 and 7, but only a few at pH 6 and 8 (Fig. 4). The light regime had a significant effect on pycnidial production and significantly less pycnidia cmx2 were produced under complete darkness as compared to 8, 12, 16 and 24 h. For all isolates after 14 d incubation, maximum pycnidial production was under continuous light (Fig. 5). Under complete darkness, isolate IMI 294735 produced significantly more pycnidia cmx2 than the other isolates (P<0.0001) (Fig. 5). Under regimes of
8 and 12 h light dx1, P130A and P176A produced less pycnidia cmx2 than the other two isolates (P<0.0001) (Fig. 5). For all isolates after both 24 and 40 h incubation, temperature had a significant effect on percentage spore germination (P<0.0001). Spore germination began after 16 h at 10 x for IMI 294735 and at 15 x for the other isolates. Spores of P176A and IMI 294735 germinated significantly faster than spores of the other two isolates (Fig. 6). Almost all spores of P176A (93 %) and IMI 294735 (90 %) germinated after 16 h at 20 x, as compared to only 22 and 24 % for P130A and DAOM 198536 (Fig. 6). Similar effects of temperature on spore germination were observed after 24 h, with a delay in germination for isolates P130A and DAOM 198536 (Fig. 6). The pH of the culture medium had a significant effect on spore germination for all isolates (P<0.0001). After 16 h of incubation, spores of P130A and DAOM 198536 germinated at pH 4–5 but were completely inhibited at pH 6 and above (Fig. 7). At pH 4, percentage spore germination was 55 and 84 %, for P176A and IMI 294735, respectively, but increased with increasing pH until pH 6 and dropped at pH 7. After 24 h incubation, spore germination of P130A and DAOM 198536 was almost completely inhibited above pH 6. Optimum germination was observed at pH 5, with percent spore
Microsphaeropsis : biocontrol and environmental factors
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Fig. 8. Effect of temperature on the number of pycnidia produced on apple leaf discs after 15 d incubation for four isolates of Microsphaeropsis. Bars with the same letters are not significantly different according to the least significant difference test (at the Pf0.05 level of confidence).
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germinations of 72 and 91%, respectively. For P176A and IMI 294735 percentage spore germination was optimal at pH 4–6 with averages around 99 %, spore germination then decreased to 58–9%, at pH 6–8. Effect of temperature and light on pycnidial production on apple leaf discs Homogeneity of variance tests indicated that data from both experimental runs could be pooled (P>0.05), hence the analysis was conducted on pooled data. Temperature had a significant effect on pycnidial production (P<0.0001). Overall, after 15 d incubation, less pycnidia cmx2 were produced on leaf discs than on culture media and greater differences amongst the isolates were observed on leaf discs (Figs 3, 8). At 5 x, none of the isolates produced pycnidia on apple leaf discs (Fig. 8). At 10 x, P176A and IMI 294735 produced significantly more pycnidia cmx2 than the other two isolates. The optimum temperature for pycnidial production was 25 x for P130A and IMI 294735, 20 x for DAOM 198536, and 15–20 x for P176A (Fig. 8). The light regime had a significant effect on pycnidial production (P<0.0001). For all light regimes, less pycnidia cmx2 were produced on leaf discs than on culture media (Figs 4, 9). For all isolates, after 15 d incubation, few pycnidia were produced under complete darkness. For most light regimes, P176A produced significantly
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more pycnidia than the others (P<0.0001) and pycnidial production increased with increasing light duration. For the other isolates, similar amounts of pycnidia were produced under 8, 12, 16 and 24 h light dx1 (Fig. 9). DISCUSSION All four isolates reduced V. inaequalis ascospore production by 52–95 % compared to the control. P130A showed the strongest potential as a biocontrol agent, followed by DAOM 198536. These observations are in accordance with previously reported in vitro screens with P130A (Philion et al. 1997, Carisse et al. 2000), but this is the first time isolates of Microsphaeropsis have been compared. Burr et al. (1996), Andrews, Berbee & Nordheim (1983), Cullen, Berbee & Andrews (1984), and Ouimet, Carisse & Neumann (1997) looked at the
O. Carisse and J. Bernier possibility of developing a biofungicide against apple scab leaf infection. This approach is fraught with several limitations, mainly because it is more difficult to attack V. inaequalis in its parasitic than in its saprophytic phase. MacHardy et al. (2001) confirmed the importance of sanitation in apple scab management and demonstrated that it is possible to delay the fungicide spray programme up to the pink stage, when the inoculum potential is very low (MacHardy, Gadoury & Rosenberger 1993). We can thus envision an autumn treatment or an early spring treatment of apple leaves with an antagonist that will interfere with the pathogen’s overwintering cycle and ascospore maturation. M. ochracea was recently identified as a potential biocontrol agent of apple scab (Carisse et al. 2000) ; however, only one isolate was tested and its environmental requirements are not known. This study identified the biological potential and some of the environmental requirements of four species of Microsphaeropsis for growth, pycnidial production and spore germination. Basic ecology of M. ochracea, M. arundinis and other species is uncertain and the information reported in the literature is sparse. To our knowledge, the only other species with known biocontrol properties is M. centaureae, a parasite of sclerotia of Sclerotinia sclerotiorum (Watson & Miltimore 1975). On the other hand, there is information on the biocontrol agent Coniothyrium minitans, which is closely related to the genus Microsphaeropsis (Sutton 1980, Whipps & Gerlagh 1992). On culture media, the optimum temperature for mycelial growth was 25 x, with a minimum of 10 x. This is slightly higher than the temperature requirement of C. minitans. At 20 x, the growth rates of P130A and DAOM 198536 were 2.3 and 2.8 mm dx1 respectively, and 3.7 and 3.9 mm dx1 for P176A and IMI 294735, respectively. The growth rate reported at this temperature for C. minitans is 3.0 mm dx1 (Campbell 1947, Turmer & Tribe 1976). Overall, more pycnidia were produced on culture media than on leaf discs, and greater differences amongst the isolates occurred on leaf discs. For example, there was not much difference in pycnidial production between all four isolates at 15–25 x on culture media. On leaf discs, the production of pycnidia of P130A and DAOM 198536 increased with temperature. P176A produced few pycnidia at 10 x on culture media, but on leaf discs their production at 10 x was only slightly less than at higher temperatures. Regardless of the media (culture or leaf disc), pycnidial production was inhibited by darkness and a minimum of 8 h light dx1 was necessary. McQuilken, Budge & Whipps (1997) reported similar results with C. minitans, which produce pycnidia in darkness, but with pycnidial production enhanced by increasing light duration. Tests on apple leaf discs are probably more representative of what occurs in nature when the biocontrol agent is applied on senescent apple leaves, than tests on culture media. However, the influence of leaf senescence, which is related to the timing of application, will have to be considered, as several biochemical changes occur during leaf senescence,
1461 including a reduction in protein, sugars and total nitrogen content (Spencer & Titus 1972). In this study, inoculation of leaf discs was on the upper surface, and pycnidia were produced only on that surface. The Microsphaeropsis isolates were probably able to directly penetrate the leaf discs, because the stomata on apple leaves are located only on the lower surface (Spencer & Titus 1973). The response of spore germination to temperature and pH clearly showed the similarity between P130A and DAOM 198536, and their distinction from the other two isolates. Germination increased with increasing temperature, but P130A and DAOM 198536 required more time to germinate. Furthermore, these isolates were strongly affected by pH, their spores germinating only at pH 4–6. This pH range is narrower than reported for C. minitans (McQuilken et al. 1997). In preliminary tests, we determined that the pH of senescent leaf discs was approximately 4.5, indicating that pH may not be a limiting factor on senescent apple leaves. From this study, it became evident that the optimum temperature for growth, pycnidial production and spore germination was 20–25 x, the optimum pH approximately 4–5 and that light was required for both pycnidial production and spore germination. Optimum conditions for growth, reproduction and germination of the biocontrol agent are not necessarily those for best biocontrol activity. For example, McQuilken et al. (1997), showed that on culture media, the optimum temperature for growth of C. minitans was 20 x and that growth was inhibited at 30 x. Later, Tu (1999) showed that maximum destruction of sclerotia of S. sclerotiorum was achieved at 30 x, provided that soil moisture was 30 % and the C. minitans inoculum was at 106 spores mlx1. P130A and DAOM 198536 were the most promising candidates for biocontrol of the teleomorph of V. inaequalis. For mass production of inoculum, temperature should not be a problem, as pycnidia were produced on culture media at 15–25 x. If spores are to be used as inoculum, light may be a problem for large containers, as a minimum of 8 h light dx1 was required for pycnidial production. Orchard application of these agents should be made when the temperature is above 15 x for at least 24 h and preferably on senescent leaves. Spring treatments under optimum temperature for spore germination and mycelial growth should also be considered in areas where that is feasible. The information acquired in this study serves as a basis for the development of these biocontrol agents. However, information on the relationship between Microsphaeropsis spp. and apple leaves, and between Microsphaeropsis spp. and V. inaequalis, will be necessarily to optimize the timing of application.
REFERENCES Andrews, J. H., Berbee, F. M. & Nordheim, E. V. (1983) Microbial antagonism to the imperfect stage of the apple scab pathogen, Venturia inaequalis. Phytopathology 73: 228–234.
Microsphaeropsis : biocontrol and environmental factors Benyagoub, M., Benhamou, N. & Carisse, O. (1998) Cytochemical investigation of the antagonistic interaction between a Microsphaeropsis sp. (isolate P130A) and Venturia inaequalis. Phytopathology 88: 605–613. Bernier, J., Carisse, O. & Paulitz, T. C. (1996) Fungal communities isolated from dead apple leaves from orchards in Quebec. Phytoprotection 77 : 129–134. Burr, T. J., Matteson, M. C., Smith, C. A., Corral-Garcia, M. R. & Huang, T.-C. (1996) Effectiveness of bacteria and yeasts from apple orchards as biological control agents of apple scab. Biological Control 6: 151–157. Campbell, W. A. (1947) A new species of Coniothyrium parasitic on sclerotia. Mycologia 39: 190–195. Carisse, O. & Bernier, J. (2002) Microsphaeropsis ochracea sp. nov. associated with dead apple leaves. Mycologia 94: 297–301. Carisse, O. & Dewdney, M. (2002) A review of non-fungicidal approaches for the control of apple scab. Phytoprotection 83: 1–29. Carisse, O., Philion, V., Rolland, D. & Bernier, J. (2000) Effect of fall application of fungal antagonists on spring ascospore production of apple scab pathogen, Venturia inaequalis. Phytopathology 90: 31–37. Cullen, D., Barbee, F. M. & Andrews, J. H. (1984) Chaetomiun globosum antagonizes the apple scab pathogen, Venturia inaequalis, under field conditions. Canadian Journal of Botany 62: 1814–1818. Dhingra, O. D. & Sinclair, J. B. (1985) Basic Plant Pathology Methods. CRC Press, Boca Raton. Heye, C. C. (1982) Biological control of the perfect stage of the apple scab pathogen, Venturia inaequalis (Cke) Wint. PhD thesis, University of Wisconsin, Madison. MacHardy, W. E., Gadoury, D. M. & Gessler, C. (2001) Parasitic and biological fitness of Venturia inaequalis: relationship to disease management strategies. Plant Disease 85: 1036–1051. MacHardy, W. E., Gadoury, D. M. & Rosenberger, D. A. (1993) Delaying the onset of fungicide programs for the control of apple scab in orchards with low potential ascospore dose of Venturia inaequalis. Plant Disease 77 : 372–375.
1462 McQuilken, M. P., Budge, S. P. & Whipps, J. M. (1997) Effects of culture media and environmental factors on spore germination, pycnidial production and hyphal extension of Coniothyrium minitans. Mycological Research 101 : 11–17. Ouimet, A., Carisse, O. & Neumann, P. (1997) Evaluation of fungal isolates for the inhibition of the vegetative growth of Venturia inaequalis. Canadian Journal of Botany 75: 626–631. Philion, V., Carisse, O. & Paulitz, T. (1997) In vitro evaluation of fungal isolates for their ability to influence leaf rheology, production of pseudothecia, and ascospores of Venturia inaequalis. European Journal of Plant Pathology 103 : 441–452. Sivanesan, A. & Waller, J. M. (1974) Venturia inaequalis. Descriptions of Plant Pathogenic Fungi and Bacteria 401 : 1–2. Spencer, P. W. & Titus, J. S. (1972) Biochemical and enzymatic changes in apple leaf tissue during autumnal senescence. Plant Physiology 49: 746–750. Spencer, P. W. & Titus, J. S. (1973) Apple leaf senescence : leaf disc compared to attached leaf. Plant Physiology 51: 89–92. Sutton, B. C. (1980) The Coelomycetes: fungi imperfecti with pycnidia, acervuli andstromata. Commonwealth Mycological Institute, Kew. Tu, J. C. (1999) Effect of temperature and moisture on the destruction of scerotia of Sclerotinia sclerotiorum by Coniothyrium minitans. Medical Faculty Landbouww, University of Gent 64 : 411–417. Turner, G. J. & Tribe, H. T. (1976) On Coniothyrium minitans and its parasitism on Sclerotium species. Transaction of the British Mycological Society 66: 97–105. Watson, A. K. & Miltimore, J. E. (1975) Parasitism of the sclerotia of Sclerotinia sclerotiorum by Microsphaeropsis centaureae. Canadian Journal of Botany 53 : 2458–2461. Whipps, J. M. & Gerlagh, M. (1992) Biology of Coniothyrium minitans and its potential for use in disease biocontrol. Mycological Research 96: 897–907.
Corresponding Editor: N. Magan
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DOI: 10.1017/S0953756202006858 Printed in the United Kingdom.
Effect of environmental factors on growth, pycnidial production and spore germination of Microsphaeropsis isolates with biocontrol potential against apple scab
Odile CARISSE* and Julie BERNIER Horticulture Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Boulevard, St-Jean-sur-Richelieu, Quebec J3B 3E6, Canada. E-mail : [email protected] Received 21 Febuary 2002; accepted 30 September 2002.
The potential as biocontrol agents of the apple scab pathogen, Venturia inaequalis and the effect of environmental factors on growth, pycnidial production and spore germination of four isolates of Microsphaeropsis were examined on culture media and leaf discs. V. inaequalis ascospore production was reduced by 52, 77, 89 and 95 % on leaf discs treated with the isolates IMI 294735, P176A, DAOM 198536 and P130A, respectively. For all isolates, optimum temperature for mycelial growth was 25 xC. More pycnidia were produced on culture media than on leaf discs. On culture media, optimum temperature was from 15 to 25 x, while on leaf discs it varied among isolates. Pycnidial production was inhibited by darkness and a minimum of 8 h light dx1 was required. Spores of isolates P130A and DAOM 198536 germinated at temperatures above 15 x and at pH 4–5, as compared to 10 x and pH 4–8 for isolates P176A and IMI 294735. The best candidates were P130A and DAOM 198536 and optimum conditions for growth, pycnidial production and spore germination were temperatures between 20 and 25 x, pH approx. 4 and light.
INTRODUCTION Apple scab, caused by Venturia inaequalis, is one of the most important diseases of apple and the most costly to control (Sivanesan & Waller 1974, MacHardy, Gadoury & Gessler 2001). Since the development of organic fungicides in the 1940s, fungicides became the sole means for managing this disease. Over the years, there have been several attempts to develop biocontrol strategies, but until recently, none had reached the point where it could be used commercially (Carisse & Dewbney 2002). V. inaequalis overwinters as incipient ascomata formed in apple leaf litter. In the spring, when leaf litter is wetted by rain, mature ascospores are forcibly discharged into the air. The ascospores are dispersed by wind from the ground to the newly emerged leaves. There, the ascospores germinate, penetrate and form lesions. In the autumn, V. inaequalis becomes a saprophyte and overwinters as ascomata initials. The most promising approach to apple scab biocontrol consists in applying a biocontrol agent in the autumn, that will interrupt overwintering of the teleomorph (MacHardy et al. 2001, Carisse & Dewdney 2002). Bernier, Carisse & Paulitz (1996) isolated 189 fungi from dead apple leaves and Philion, Carisse & Paulitz * Corresponding author.
(1997) screened some of these for their ability to inhibit ascospores production in in vitro tests. From this evaluation, five isolates, including two Microsphaeropsis (P130A and P176A), were selected. These potential biocontrol agents were further tested under orchard conditions with the most reliable reduction in ascospore production being obtained with isolate P130A (Carisse et al. 2000), described as the new species M. ochracea (Carisse & Bernier 2002). Based on a study of the interaction between M. ochracea and V. inaequalis (Benyagoub, Benhamou & Carisse 1998), it was assumed that M. ochracea must penetrate the leaves in order to interact with the formation of ascomata by V. inaequalis. However, information on the ecology of Microsphaeropsis species is sparse, and non-existent for M. ochracea. It is not clear whether M. ochracea or other Microsphaeropsis species colonize and penetrate the apple leaves and produce pycnidia when applied in the autumn on senescent apple leaves, either on the tree canopy (after harvest but before leaf fall) or on the ground. The potential of Microsphaeropsis isolates to reduce ascospore production by V. inaequalis has been demonstrated under controlled and small plot conditions (Carisse et al. 2000). For commercial uses, proper timing of the antagonist is crucial. The autumn treatment should be made so that all leaves are treated and to
Microsphaeropsis : biocontrol and environmental factors favour colonization by the antagonist in order to maximize the possibility of contact between the antagonist and V. inaequalis. If the antagonist is applied on the tree canopy before leaf fall (soon after harvest) and if commercial sprayers are used, most leaves should be sprayed but the environment at the leaf level may not favour leaf colonization by the antagonist or allow it to maintain its population at a high level. On the other hand, if the antagonist is applied on the ground after leaf fall, all leaves may not be reached and weather conditions in northern regions may be unfavourable to antagonistic activity. Before deciding which spray strategy is most promising, it is important to determine the effect of the environment on fungal growth, pycnidial production, and spore germination. The objective of this study was to compare the potential of four Microsphaeropsis spp. as biocontrol agents of apple scab and to obtain quantitative information on the effect of temperature, pH and light on fungal growth, pycnidial production and spore germination of M. ochracea and three related species.
MATERIALS AND METHODS Source of isolates and inoculum production Four isolates, designated as M. ochracea P130A, Microsphaeropsis sp. P176A, Microsphaeropsis sp. DAOM 198536 and M. arundinis IMI 294735 were included in this study. The first two isolates were isolated from dead apple leaves collected in Quebec orchards (Bernier et al. 1996) and stored in water or soil at 4 xC ; P130A is deposited at ATCC 74412. The other two isolates also originated from apple leaves or fruits and were obtained from the Canadian Fungal Culture Collection (DAOM, Ottawa) and the CABI Bioscience UK Centre (IMI, Egham). John Andrews (Wisconsin University) kindly provided the culture of Athelia bombacinai, which was used as a positive control (Heye 1982). Effect of isolates on ascospore productions Disease-free apple leaves were collected from Malus pumila cv. ‘ McIntosh’ trees. Leaf discs 2.6 cm diam were cut and sterilized by irradiation (10 h under 40 kGy [4 Mrad] gamma radiation). Petri dishes 9 cm diam were filled with 20 ml of autoclaved PerliteTM (Perlite Atlas, Beauport, QC) and 10 ml of sterilized distilled water. The irradiated leaf discs were placed in the Petri dish on perlite (2 discs per dish). A mycelial suspension of V. inaequalis was made from a mixture of ten isolates, all originating from scabbed leaves. A mixture of isolates was used to increase the number of pairs of compatible mating types, and consequently ensure the production of ascomata. Each isolate was previously grown on PDA at 18 x. As the growth rate of each isolate was quite different, when the isolates covered more than twothirds of the surface of a 9 cm Petri dish they were stored at 2 x until inoculation of the leaf discs (max. 2 wk). To
1456 prepare the mixture of V. inaequalis isolates, the content of one Petri dish of each isolate was placed in a sterile plastic bag (Steward Medical, London) and 200 ml of sterile water was added. The content of the bag was homogenized in a Stomacher TM laboratory blender (Seward Medical) for 6 min. Each leaf disc was inoculated with 150 ml of the V. inaequalis mycelial slurry. Immediately after the disc inoculation with V. inaequalis, the discs were inoculated with the four isolates of Microsphaeropsis sp. at a rate of 100 ml of a spore suspension of 4.5r108 spores mlx1. Athelia bombacina (positive control) was stored on PDA slants under mineral oil maintained at 2 x. Prior to the disc inoculation, the fungus was cultured on PDA and mycelial slurry was made from 3-wk-old cultures as described for V. inaequalis. Each disc was inoculated with A. bombacina at a rate of 100 ml of mycelial slurry per leaf disc. For each isolate, eight Petri dishes were inoculated and the control consisted of eight dishes of leaf discs inoculated only with V. inaequalis. The experiment was set up as a completely randomized design with eight repetitions and the entire experiment was conducted twice. Several supplementary controls were prepared and used to follow the maturation of ascomata and to determine the optimal time for ascospore extraction. The leaf discs were incubated at room temperature for 3 wk, then transferred to a cold room adjusted to 4 x with 90% relative humidity in complete darkness for 4 wk, followed by 12 wk at 10 x, with 90 % relative humidity and complete darkness. When ascomata were mature in the controls, all dishes were brought to the laboratory and kept at room temperature (20–25 x) for another 3 wk to favour ascospore maturation. To evaluate ascospore production per leaf disc, two discs were placed in a 50 ml Falcon test tube filled with 20 ml of distilled water. A bubbler type apparatus, developed by Philion et al. (1997) was used to force ascospore ejection into the water. Air was forced at the rate of 25.5 l minx1 into the Falcon tubes through Pasteur pipettes for a total of 6 h. The aliquot of each tube was decanted into another tube and a few drops of Lugol’s iodine solution were added to each ascospore suspension to avoid germination. The contents of the tubes were centrifuged at 3800 rev minx1 for 10 min (Accuspin FR, Beckman, CA). The liquid was carefully drawn out of the tubes and 5 ml was conserved so that the ascospore pellet and surrounding liquid were not disturbed. Ascospore concentration was estimated using a haemocytometer and ascospore productivity reported as ascospores cmx2 of leaf disc. Effect of temperature on mycelial growth on culture media The inoculum of Microsphaeropsis isolates was produced by growing them on potato dextrose agar (PDA ; Difco, MD) at room temperature for 2 wk. The spore suspensions were made by scraping and transferring pycnidia into a 0.85 % saline solution. The suspensions
O. Carisse and J. Bernier were adjusted to 1r106 spores mlx1 using a haemocytometer. The effect of temperature on mycelial growth was evaluated on PDA culture media. A mycelial plug 6 mm diam was removed from the periphery of an actively growing culture and placed in the centre of a PDA dish. Dishes were incubated at different temperatures and radial growth was measured after 1 and 2 wk of incubation. For each fungus, four dishes were inoculated and the experiment was conducted twice. Effect of temperature, pH and light on pycnidial production and of temperature and pH on spore germination on culture media To study pycnidial production on culture media, 50 ml of spore suspension was inoculated on PDA Petri dishes and spread onto the entire surface of each dish using a glass rod. For each treatment, four Petri dishes were inoculated. To test the effect of temperature, dishes were incubated for 3 d at room temperature (20–25 x) to allow hyphal growth prior to transfer to controlled-environment growth chambers (Percival, Bonne IW ; model I-30BLLX) adjusted to temperatures of 5, 10, 15, 20 and 25 x (¡0.5 x) with a photoperiod of 12 h light dx1 provided by incandescent and fluorescent lights (Vita Lite, Duro-Test, Montreal). The light was adjusted to 1950–80 lx in all incubators using a LI-1000 data logger (LI-COR, NE). To test the effect of pH, PDA medium was buffered prior to autoclaving with a citrate-phosphate solution to obtain pH values of 4, 5, 6 and 7, and with a phosphate buffer solution to obtain a pH of 8. When adjusting the medium to pH 4, care was taken to avoid acid hydrolysis, which prevents gelation of agar following cooling. The citrate-phosphate buffer solution and the PDA medium were autoclaved separately and then aseptically mixed together during cooling. The buffer solutions were prepared following the method in Dhingra & Sinclair (1985). The pH was measured at the time of inoculation of dishes and at the end of the experiment to make sure that it had been maintained throughout the experiment. The inoculated dishes were incubated in controlled-environment growth chambers adjusted to 20 x with a photoperiod of 12 h light as described above. To test the effect of light regime, the inoculated dishes were incubated at 20 x under daylight of 0, 8, 12, 16 and 24 h provided by fluorescent and incandescent lights (as described above). The same protocol as described above was used to study the effect of temperature and light regime on spore germination on culture media with the following modifications: the spore suspensions were spread onto water agar (WA ; Difco, MD), 15 g lx1, instead of PDA. Secondly, the dishes were transferred to the growth chambers adjusted to different temperatures immediately after inoculation and five replicates were used per treatment. To evaluate pycnidial production, the inoculated dishes were removed from the incubators and plugs
1457 3 mm diam ; two plugs per plate, were cut at random in the fungal growth area and removed from the mycelial mat. The plugs were transferred to 500 ml of sterile distilled water in 2 ml Eppendorf tubes placed in boiling water for 15 min to melt the agar. The mycelial mat containing the pycnidia was then transferred on a glass slide and the total number pycnidia per plug counted with a microscope at r25. Samples were collected every 2–3 d over a period of 14–18 d, depending on experiments. Spore germination was assessed after 16, 24 and 40 h of incubation using an inverse microscope adjusted to r250. A total of 100 spores from each dish were examined and a conidium was considered germinated when the germ tube was at least the length of the spore. Effect of temperature and light on pycnidial production on apple leaf discs The effects of temperature and light on pycnidial production were examined on apple leaf discs. Leaf discs 2.6 cm diam were cut from green apple leaves, cv. McIntosh, using a cork borer. The discs were sterilized by irradiation (10 h under 40 kGy [4 Mrad] gamma radiation). Irradiated leaf discs were stored at 4 x in hermetically closed glass jars until needed for the experiments. To provide a moist environment but to avoid the contact between the fungi and the media, the leaf discs were placed on previously autoclaved wood coffee sticks placed on the surface of WA dishes with two discs per dish. For the four isolates, spore suspensions were prepared as previously described and 100 ml of suspension was inoculated on each leaf disc. To test the effect of temperature, the dishes were incubated for 3 d at room temperature to allow hyphal growth prior to transfer to the controlled-environment growth chambers adjusted to different temperatures with light adjusted to 1720–1885 lx. To test the effect of light regime, the inoculated dishes were incubated at 20 x under daylight of 0, 8, 12, 16 and 24 h provided by fluorescent and incandescent light. The density of pycnidia on each leaf disc was evaluated by counting the number of pycnidia in two binocular fields, which corresponded to an area of 1.36 cm2 (25.52 % of the surface of the disc). Observations were made every 2–3 d over a period of 13–15 d depending on the experiment. Statistical analysis All experiments were conducted following a completely randomized block design with 4–8 blocks (dishes), depending on the experiment, and were conducted twice. An F-test was used to determine if the variances of the two experimental runs were homogenous and if data could be pooled. Analysis of variance was used to test the effect of fungal isolates on ascospore production and of temperature, pH and light regime on mycelial growth, pycnidial production and spore germination. Significant
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Effect of isolates on ascospore production The homogeneity of variance test indicated that data from both experimental runs could be pooled (P>0.05) ; hence the analysis was conducted on pooled data. All Microsphaeropsis isolates and A. bombacina significantly reduced ascospore production (P<0.0001) (Fig. 1). An average of 1146.54 ascospores cmx2 of leaf disc was produced on the untreated discs (V. inaequalis only) and this amount was reduced by 51.99, 77.36, 88.86, 93.71 and 95.18 % on leaf discs treated with the isolates IMI 294735, P176A, DAOM 198536, A. bombacina and P130A, respectively. There was no significant difference in ascospore production on leaf discs treated with A. bombacina and Microsphaeropsis P130A or DAOM 198536. Effect of temperature on mycelial growth on culture media Homogeneity of variance tests indicated that data from both experimental runs could be pooled (P>0.05), hence the analysis was conducted on pooled data. For all isolates, after 14 d incubation, mycelial growth increased with increasing temperature following a similar pattern (Fig. 2). Temperature had a significant effect on mycelial growth of all Microsphaeropsis isolates (P<0.0001). At 5 x, there was no significant difference between growth in mm for the four isolates (Fig. 2). At temperatures above 5 x, isolates P176A and IMI 294735 grew significantly more than the other two isolates
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(P<0.0001) and at temperatures of 15 and 25 x isolate IMI 294735 showed significantly higher growth than P176A (P<0.0001) (Fig. 2). Effect of temperature, pH and light on pycnidial production and of temperature and pH on spore germination on culture media For all tests, variances were homogenous (P>0.05), hence the analysis was conducted on pooled data. Overall, temperature had a significant effect on pycnidial production cmx2 (P<0.0001). For all isolates after 15 d of incubation, only few pycnidia were produced at 5 x and isolate IMI 294735 produced significantly more pycnidia cmx2 than the other isolates (Fig. 3). At 10 x, DAOM 198536 and IMI 294735 produced significantly more pycnidia cmx2 than the other isolates (Fig. 3). At temperatures of 15, 20 and 25 x, there was no significant
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Fig. 5. Effect of light regime on the number of pycnidia produced on PDA after 14 d incubation for four isolates of Microsphaeropsis. Bars with the same letters are not significantly different according to the least significant difference test (at the Pf0.05 level of confidence).
difference between the numbers of pycnidia produced by the four isolates (P<0.0001) (Fig. 3). Overall, pH had a significant effect on pycnidial production cmx2 (P<0.0001) but large variations were observed among observations. For all isolates after 16 d incubation, the optimum for pycnidial production was pH 4, and for most isolates only a few pycnidia were produced above pH 4. IMI 294735 differed from the other isolates producing a large number of pycnidia cmx2 at pH 5 and 7, but only a few at pH 6 and 8 (Fig. 4). The light regime had a significant effect on pycnidial production and significantly less pycnidia cmx2 were produced under complete darkness as compared to 8, 12, 16 and 24 h. For all isolates after 14 d incubation, maximum pycnidial production was under continuous light (Fig. 5). Under complete darkness, isolate IMI 294735 produced significantly more pycnidia cmx2 than the other isolates (P<0.0001) (Fig. 5). Under regimes of
8 and 12 h light dx1, P130A and P176A produced less pycnidia cmx2 than the other two isolates (P<0.0001) (Fig. 5). For all isolates after both 24 and 40 h incubation, temperature had a significant effect on percentage spore germination (P<0.0001). Spore germination began after 16 h at 10 x for IMI 294735 and at 15 x for the other isolates. Spores of P176A and IMI 294735 germinated significantly faster than spores of the other two isolates (Fig. 6). Almost all spores of P176A (93 %) and IMI 294735 (90 %) germinated after 16 h at 20 x, as compared to only 22 and 24 % for P130A and DAOM 198536 (Fig. 6). Similar effects of temperature on spore germination were observed after 24 h, with a delay in germination for isolates P130A and DAOM 198536 (Fig. 6). The pH of the culture medium had a significant effect on spore germination for all isolates (P<0.0001). After 16 h of incubation, spores of P130A and DAOM 198536 germinated at pH 4–5 but were completely inhibited at pH 6 and above (Fig. 7). At pH 4, percentage spore germination was 55 and 84 %, for P176A and IMI 294735, respectively, but increased with increasing pH until pH 6 and dropped at pH 7. After 24 h incubation, spore germination of P130A and DAOM 198536 was almost completely inhibited above pH 6. Optimum germination was observed at pH 5, with percent spore
Microsphaeropsis : biocontrol and environmental factors
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Fig. 8. Effect of temperature on the number of pycnidia produced on apple leaf discs after 15 d incubation for four isolates of Microsphaeropsis. Bars with the same letters are not significantly different according to the least significant difference test (at the Pf0.05 level of confidence).
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Fig. 7. Effect of pH on mean spore germination on PDA after 16 and 24 h incubation for four isolates of Microsphaeropsis (standard errors 25.58 and 29.64 after 16 and 24 h incubation, respectively).
germinations of 72 and 91%, respectively. For P176A and IMI 294735 percentage spore germination was optimal at pH 4–6 with averages around 99 %, spore germination then decreased to 58–9%, at pH 6–8. Effect of temperature and light on pycnidial production on apple leaf discs Homogeneity of variance tests indicated that data from both experimental runs could be pooled (P>0.05), hence the analysis was conducted on pooled data. Temperature had a significant effect on pycnidial production (P<0.0001). Overall, after 15 d incubation, less pycnidia cmx2 were produced on leaf discs than on culture media and greater differences amongst the isolates were observed on leaf discs (Figs 3, 8). At 5 x, none of the isolates produced pycnidia on apple leaf discs (Fig. 8). At 10 x, P176A and IMI 294735 produced significantly more pycnidia cmx2 than the other two isolates. The optimum temperature for pycnidial production was 25 x for P130A and IMI 294735, 20 x for DAOM 198536, and 15–20 x for P176A (Fig. 8). The light regime had a significant effect on pycnidial production (P<0.0001). For all light regimes, less pycnidia cmx2 were produced on leaf discs than on culture media (Figs 4, 9). For all isolates, after 15 d incubation, few pycnidia were produced under complete darkness. For most light regimes, P176A produced significantly
30 Pycnidia cm–2 d–1
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Fig. 9. Effect of light regime on the number of pycnidia produced on apple leaf discs after 15 d incubation for four isolates of Microsphaeropsis. Bars with the same letters are not significantly different according to the least significant difference test (at the Pf0.05 level of confidence).
more pycnidia than the others (P<0.0001) and pycnidial production increased with increasing light duration. For the other isolates, similar amounts of pycnidia were produced under 8, 12, 16 and 24 h light dx1 (Fig. 9). DISCUSSION All four isolates reduced V. inaequalis ascospore production by 52–95 % compared to the control. P130A showed the strongest potential as a biocontrol agent, followed by DAOM 198536. These observations are in accordance with previously reported in vitro screens with P130A (Philion et al. 1997, Carisse et al. 2000), but this is the first time isolates of Microsphaeropsis have been compared. Burr et al. (1996), Andrews, Berbee & Nordheim (1983), Cullen, Berbee & Andrews (1984), and Ouimet, Carisse & Neumann (1997) looked at the
O. Carisse and J. Bernier possibility of developing a biofungicide against apple scab leaf infection. This approach is fraught with several limitations, mainly because it is more difficult to attack V. inaequalis in its parasitic than in its saprophytic phase. MacHardy et al. (2001) confirmed the importance of sanitation in apple scab management and demonstrated that it is possible to delay the fungicide spray programme up to the pink stage, when the inoculum potential is very low (MacHardy, Gadoury & Rosenberger 1993). We can thus envision an autumn treatment or an early spring treatment of apple leaves with an antagonist that will interfere with the pathogen’s overwintering cycle and ascospore maturation. M. ochracea was recently identified as a potential biocontrol agent of apple scab (Carisse et al. 2000) ; however, only one isolate was tested and its environmental requirements are not known. This study identified the biological potential and some of the environmental requirements of four species of Microsphaeropsis for growth, pycnidial production and spore germination. Basic ecology of M. ochracea, M. arundinis and other species is uncertain and the information reported in the literature is sparse. To our knowledge, the only other species with known biocontrol properties is M. centaureae, a parasite of sclerotia of Sclerotinia sclerotiorum (Watson & Miltimore 1975). On the other hand, there is information on the biocontrol agent Coniothyrium minitans, which is closely related to the genus Microsphaeropsis (Sutton 1980, Whipps & Gerlagh 1992). On culture media, the optimum temperature for mycelial growth was 25 x, with a minimum of 10 x. This is slightly higher than the temperature requirement of C. minitans. At 20 x, the growth rates of P130A and DAOM 198536 were 2.3 and 2.8 mm dx1 respectively, and 3.7 and 3.9 mm dx1 for P176A and IMI 294735, respectively. The growth rate reported at this temperature for C. minitans is 3.0 mm dx1 (Campbell 1947, Turmer & Tribe 1976). Overall, more pycnidia were produced on culture media than on leaf discs, and greater differences amongst the isolates occurred on leaf discs. For example, there was not much difference in pycnidial production between all four isolates at 15–25 x on culture media. On leaf discs, the production of pycnidia of P130A and DAOM 198536 increased with temperature. P176A produced few pycnidia at 10 x on culture media, but on leaf discs their production at 10 x was only slightly less than at higher temperatures. Regardless of the media (culture or leaf disc), pycnidial production was inhibited by darkness and a minimum of 8 h light dx1 was necessary. McQuilken, Budge & Whipps (1997) reported similar results with C. minitans, which produce pycnidia in darkness, but with pycnidial production enhanced by increasing light duration. Tests on apple leaf discs are probably more representative of what occurs in nature when the biocontrol agent is applied on senescent apple leaves, than tests on culture media. However, the influence of leaf senescence, which is related to the timing of application, will have to be considered, as several biochemical changes occur during leaf senescence,
1461 including a reduction in protein, sugars and total nitrogen content (Spencer & Titus 1972). In this study, inoculation of leaf discs was on the upper surface, and pycnidia were produced only on that surface. The Microsphaeropsis isolates were probably able to directly penetrate the leaf discs, because the stomata on apple leaves are located only on the lower surface (Spencer & Titus 1973). The response of spore germination to temperature and pH clearly showed the similarity between P130A and DAOM 198536, and their distinction from the other two isolates. Germination increased with increasing temperature, but P130A and DAOM 198536 required more time to germinate. Furthermore, these isolates were strongly affected by pH, their spores germinating only at pH 4–6. This pH range is narrower than reported for C. minitans (McQuilken et al. 1997). In preliminary tests, we determined that the pH of senescent leaf discs was approximately 4.5, indicating that pH may not be a limiting factor on senescent apple leaves. From this study, it became evident that the optimum temperature for growth, pycnidial production and spore germination was 20–25 x, the optimum pH approximately 4–5 and that light was required for both pycnidial production and spore germination. Optimum conditions for growth, reproduction and germination of the biocontrol agent are not necessarily those for best biocontrol activity. For example, McQuilken et al. (1997), showed that on culture media, the optimum temperature for growth of C. minitans was 20 x and that growth was inhibited at 30 x. Later, Tu (1999) showed that maximum destruction of sclerotia of S. sclerotiorum was achieved at 30 x, provided that soil moisture was 30 % and the C. minitans inoculum was at 106 spores mlx1. P130A and DAOM 198536 were the most promising candidates for biocontrol of the teleomorph of V. inaequalis. For mass production of inoculum, temperature should not be a problem, as pycnidia were produced on culture media at 15–25 x. If spores are to be used as inoculum, light may be a problem for large containers, as a minimum of 8 h light dx1 was required for pycnidial production. Orchard application of these agents should be made when the temperature is above 15 x for at least 24 h and preferably on senescent leaves. Spring treatments under optimum temperature for spore germination and mycelial growth should also be considered in areas where that is feasible. The information acquired in this study serves as a basis for the development of these biocontrol agents. However, information on the relationship between Microsphaeropsis spp. and apple leaves, and between Microsphaeropsis spp. and V. inaequalis, will be necessarily to optimize the timing of application.
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Corresponding Editor: N. Magan