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Department of Plant Pathology, Washington State University, Tree Fruit Research and Extension Center, 1100 North Western Avenue, Wenatchee, Washington 98801
J.D. Rogers
Department of Plant Pathology, Washington State University, Pullman, Washington 99164–6430
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sphaeropsis pyriputrescens, the causal agent of Sphaeropsis rot of pears and apples, is a recently described species. In this study the effects of culture media, temperature, water potential, pH and light on mycelial growth and pycnidial production of S. pyriputrescens were evaluated. Apple juice agar and pear juice agar were most suitable for mycelial growth of all six isolates tested. Cornmeal agar was not suitable for either mycelial growth or pycnidial production. The fungus grew from –3 to 25 C, with optimum growth at 20 C and no growth at 30 C. The fungus grew at water potential as low as –5.6 MPa on potassium chloride-amended potato-dextrose agar (PDA). Hyphal extension was not observed at –7.3 MPa after 10 d incubation, but growth resumed when the inoculum plugs were placed on PDA. The fungus grew at pH 3.3–6.3 and optimum growth was at pH 3.3–4.2. No mycelial growth was observed at pH above 7.2 after 10 d incubation, but growth resumed when the inoculum plugs were transferred onto PDA. Regardless of medium tested, few pycnidia formed at 20 C in the dark. Pycnidial production was enhanced significantly by fluorescent light, but continuous light appeared to reduce pycnidial production, depending on the medium. Oatmeal agar (OMA) was most suitable for production of pycnidia and conidia. Pycnidia that formed on 3 wk old OMA cultures at 20 C under 12 h light/12 h dark produced abundant conidia, and the technique is recommended for inoculum production.
Key words: fungal physiology, postharvest pathogenic fungi
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Xiao et al 2004). The disease was found first on d’Anjou pears (Pyrus communis L.), and later it was determined that Sphaeropsis rot can cause even more serious problems on apples (Malus x domestica Borkh.). In one case 24% of the apples in storage bins was rotted by this fungus after several months of storage. A total of 194 bins (ca. 400 kg of fruit per bin) from one orchard had to be discarded. Despite description of the fungus, little information is available on its biology, including environmental requirements for growth and sporulation (Xiao and Rogers 2004). This information will be valuable to further mycological and pathological research on the fungus and disease, including development of measures for disease management. The objective of this study was to provide information on effects of culture media and various environmental factors including temperature, pH, water potential and light on mycelial growth and pycnidial production of S. pyriputrescens. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Inoculum and inoculation.— – Inoculum and inoculation of plates were prepared as follows unless otherwise specified. Isolates were grown on PDA at 20 C in the dark for 4 d. Four mm diam agar plugs were removed with a sterile cork borer from the leading edges of colonies, and one such plug was placed in the center of each 90 mm Petri plate containing a medium. Plates then were wrapped with Parafilm (Pechiney Plastic Packaging, Chicago, Illinois).
Effect of culture media on mycelial growth.— –
Mycelial growth of six isolates of S. pyriputrescens was evaluated on eight media (TABLE I). PDA and cornmeal agar (CMA) were from Difco Laboratories (Franklin Lakes, New Jersey) and prepared according to label directions. The recipes for other media follow. Malt-extract agar (MEA): 20 g Difco malt extract and 15 g agar in 1 L of de-ionized water. Oatmeal agar (OMA): 60 g of iron- and zinc-fortified single-grain oatmeal (Gerber, Fremont, Michigan) with 15 g of agar in 1 L of de-ionized water. Czapek-Dox agar (CDA): 2 g NaNO3, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, and 0.01 g FeSO4·7H2O in 1L of de-ionized water in a water bath for 15 min. After cooling, 30 g sucrose and 15 g agar were added. V-8 juice agar (V8), apple juice agar (AJA) and pear juice agar (PJA) were prepared by adding 200 mL of V-8 juice (Campbell Soup Co., Camden, New Jersey), apple juice (TreeTop, Selah, Washington) and freshly made pear juice in 800 mL of de-ionized water respectively, and adding 3 g CaCO3, 15 g agar into each medium. OMA medium was autoclaved 90 min and all other media were autoclaved 35 min using a Market Forge Sterilizer (Alfa Medical, Hempstead, New York).
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Temperature on mycelial growth.— – Effect of temperature on mycelial growth was evaluated on PDA. Inoculated plates were placed in plastic bags and incubated at –3, 0, 5, 10, 15, 17, 20, 22, 25, 30 or 35 C in the dark. In each incubator (at each temperature) plates (three replicates for each isolate) were arranged in a random complete block design. Colony diameters were measured as described above at 2, 3 and 4 d after inoculation at 10, 15, 20 and 25 C. Colony diameters were measured at 4, 10 and 20 d after inoculation at –3 and 0 C, and 2, 3, 4 and 10 d after inoculation at 5, 30 and 35 C. Cultures that did not grow after 10 d of incubation at 30 and 35 C were moved to 20 C, and after 4 and 10 d of incubation these cultures were examined to determine whether the fungus resumed growth. Daily radial growth rates were calculated.
Effect of water potential on mycelial growth.— –
To determine the influence of water potential on mycelial growth, PDA was amended with potassium chloride (KCl) at various concentrations before sterilization to obtain water potentials of –0.3, –0.8, –1.2, –2.1, –3.9, –4.7, –5.6 and –7.3 MPa, according to Robinson and Stokes (1959). Inoculated plates were placed in plastic bags and incubated at 20 C in the dark. The experimental design was a random complete block with three replicate plates of each isolate for each water potential. The colony diameters were measured at 2, 3 and 4 d after inoculation, and plates at –3.9, –4.7 and –5.6 MPa also were measured at 10 and 20 d after inoculation. Daily radial growth was calculated. Mycelial plugs that had been used to inoculate –7.3 MPa plates in which no mycelial growth occurred after 10 d of incubation were transferred onto unamended PDA (–0.3 MPa) and incubated at 20 C in the dark an additional 10 d to determine whether the fungus resumed growth.
Effect of pH on mycelial growth.— –
To examine the effect of medium pH on colony growth, sterile double-strength PDA was mixed with an equal volume of the buffer to give the desired concentration of medium. The pH was obtained over the ranges 3–7 and 7–9 with citrate phosphate (0.1 M solution of citric acid, 0.2 M solution of Na2HPO4·7H2O) and Tris (hydroxymethyl) aminomethane (0.2 M solution of Tris aminomethane, 0.2 M HCl) buffers respectively (Gomori 1955). The buffer solution and PDA were autoclaved separately and aseptically mixed during cooling. The pH was measured with a pH meter (Corning 440, with 476436 electrode; Corning Inc., Corning, New York) before pouring. Plates were inoculated as described above and incubated at 20 C in the dark. The experimental design was a random complete block with three replicate plates of each isolate for each pH treatment. The colony diameters were measured at 2, 3 and 4 d after inoculation, and colonies at pH greater than 7 also were measured at 10 and 20 d after inoculation. Daily radial growth was calculated. Mycelial plugs that had been used to inoculate media with pH greater than 7, in which no mycelial growth occurred after 10 d incubation, were transferred onto unamended PDA and incubated at 20 C in the dark an additional 10 d to determine whether the fungus resumed growth.
Effect of culture media and light.— – The effect of light on mycelial growth and pycnidial production was evaluated on the same eight media used in the media-effect study. Isolate 2142 was used in this study. Inoculated plates were incubated at 20 C under three light regimes: 24 h dark, 24 h fluorescent light (5 W m–2), and 12 h light/12 h dark. Plates under the 24 h dark regime were covered with aluminum foil and placed in a cardboard box. Experiments at 24 h dark and 12 h light/12 h dark regimes were performed at the same time, and the 24-h light regime experiment was performed separately using the same incubator. There were three replicate plates for each medium under each light regime. Colony diameters were measured as described above at 2, 3 and 4 d after inoculation. Daily growth was calculated. Pycnidia development on different media was visually evaluated weekly. Three wk after inoculation, colony diameter was measured and percentage of the colony area yielding pycnidia in each plate was visually estimated. The number of pycnidia in six 1 cm diam fields in each plate was counted under a dissecting microscope at 20x. Five pycnidia were selected from the edge of the colony and from the middle of the colony radius from each plate respectively. Pycnidia were crushed gently in water on glass slides, and the presence and maturity of conidia were examined under a microscope.
An index was created to compare the efficiency of treatments for pycnidial production. Index of pycnidial production is defined as (number of pycnidia in 1 cm diam field x proportion of colony area yielding pycnidia) x (colony diam/85), where 85 is the maximum colony diameter. To quantify conidia, the agar medium from each plate containing pycnidia was blended in 200 mL water using a commercial blender (Hamilton Beach, Procter Silex Inc., Washington, North Carolina). Resulting suspensions were filtered through four layers of cheesecloth, and spore concentrations then were quantified with a hemacytometer. The number of conidia produced per plate was calculated.
Statistical analysis.— – All experiments were performed twice. An F-test was used to determine if variance of the two runs of each experiment was homogeneous and if data could be pooled. The homogeneity of variance test indicated that the data from both runs of each experiment could be pooled, and thus all further analyses were conducted on pooled data. Data from the effect of culture media on mycelial growth were subjected to analysis of variance. Data from studies on temperature effect and the effects of culture media and light on mycelial growth and pycnidial production were analyzed by split-plot analysis of variance with temperature as main plot and isolate as subplot (temperature study) or light regimes as main plot and media as subplot (pycnidial production study). All analyses were performed with PC SAS PROC GLM (version 8.2, SAS Institute, Cary, North Carolina), and mean separations were made by least significant difference (P = 0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In general, AJA and PJA were most favorable for fast radial growth of mycelium of all six isolates tested. At 20 C, colonies on these two media reached the edge of the plates after 5 d of inoculation. The fungus formed circular, colorless and compact colonies with few or no aerial hyphae. On PDA the fungus initially formed a circular colony with hyaline hyphae and 5–7 d later turned light yellow to yellow starting from the center of the plate. The colony was compact with some aerial hyphae. On OMA and V8 the fungus formed circular white colonies and the hyphae at the edge appeared aerial. On MEA and CMA the fungus formed circular, colorless colonies with no aerial hyphae and mycelial growth on CMA was poor with scanty and thin mycelium. On CDA, the fungus formed circular colonies with some aerial hyphae.
There was a significant interaction (P < 0.0001) between medium and isolate on radial mycelial growth. In other words the radial mycelial growth rates of S. pyriputrescens isolates were medium dependent. In general 1026 was the fastest growing isolate and 1890 was the slowest growing isolate on most of the media tested (TABLE I).
Effect of temperature.— –
The rate of mycelial growth of all six isolates followed similar trends in response to changes in temperature (FIG. 1). The rate of mycelial growth increased as temperature increased up to 20 C and then decreased rapidly as temperature increased. Slight changes in colony morphology were observed at –3, 0, 5 and 25 C. At –3 C, mycelial growth was relatively slow (0.28 mm d–1 in average) with fluffy, aerial mycelium. At 0, 5 and 25 C colonies sometimes were not completely circular, with thin mycelium. The average growth at these temperatures was 0.8, 2.7 and 2.5 mm d–1 respectively. Optimum growth occurred at 20 C for all six isolates, with an average growth of 7.3 mm d–1. At 30 C mycelial growth was not observed after 10 d of incubation, but the fungus resumed growth when the plates were moved to 20 C. All six isolates were not able to grow at 35 C and failed to resume growth after 10 additional d of incubation at 20 C. They presumably were dead.
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). In general isolates 1026, 2142 and 2380 grew faster than other isolates and there was no significant difference in mean growth rates among these three isolates. The slowest growing isolate was 1890.
Effect of water potential.— –
The six isolates of S. pyriputrescens followed similar trends in response to changes in water potential (FIG. 2). The rate of radial growth initially increased as water potential decreased for most isolates. The maximum growth occurred at water potential around –0.8 MPa for most isolates except for isolate 1022, which had the highest growth on unamended PDA (–0.3 MPa). The radial growth was reduced tremendously as water potential decreased from –1.2 to –3.9 MPa. The fungus grew actively at water potential of –1.2 MPa and attained an overall average of 47 mm colony diam after 4 d of incubation at 20 C. Differences in colony morphology, such as irregular growth or sectoring, grown at water potential below –2.1 MPa, were observed. The fungus grew at –5.6 MPa, but only slowly with no measurable growth after 4 d of incubation, and attained an average of 10 mm colony diam after 20 d of incubation at 20 C. All isolates that did not grow at –7.3 MPa after 10 d of incubation resumed growth after they were transferred onto unamended PDA at 20 C.
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). Optimum growth occurred at pH 3.3 or 4.2, with an overall average of 65 mm colony diam after 4 d of incubation at 20 C. A notable decline of the growth occurred when pH increased from 4.2 to 5.2. At pH 6.3, the fungus grew slowly, with an average of 23 mm colony diam after 10 d of incubation at 20 C. None of the isolates grew at pH 7.2 or above.
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). The light x medium interaction for radial growth was significant (P < 0.0001). The radial growth was significantly slower under the 12 h light/12 h dark regime than under the 24 h dark or 24 h light regime on all media, except V8 agar on which the growth rate increased as the light exposure lengthened.
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). The fungus grown on OMA started to form pycnidia in the center of the plate 7 d after inoculation. Formation of pycnidia on PDA, AJA and PJA under the 12 h light/12 h dark regime was 3 d behind that on OMA. The fungus grown on all media except on CMA started to form pycnidia after 7 d of incubation under the 24 h light regime. On PDA pycnidia were aggregated in the center of the plate under the 12 h light/12 h dark, whereas under the 24 h light regime, pycnidia more uniformly were scattered on the plate. Regardless of light, CDA produced white, fluffy aerial mycelium at the edge of the plate after 7 d of incubation. Pycnidia formed only on the fluffy mycelial masses under both 12 and 24 h light regimes. On MEA, AJA and PJA, many pycnidia were immersed in the media under both 12 h light/12 h dark and 24 h light regimes. Mature brown conidia were abundant inside pycnidia on all media, except CDA and V8 on which approximately 50% of the conidia were immature (pale) under both 12 h light/12 h dark and 24 h light regimes.
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). Fungus grown on OMA started to form a few pycnidia, but no conidia were observed inside the pycnidia. Other media did not produce pycnidia at 20 C in the dark.
There was a significant interaction (P < 0.0001) between medium and light regimes for both index of pycnidial production and conidial production (TABLE III). Under 12 h light/12 h dark, the entire colony area yielded pycnidia on OMA and both index of pycnidial production and number of conidia produced per plate were significantly higher than on other media. The index of pycnidial production and number of conidia produced per plate on OMA were significantly reduced under the 24 h continuous light regime and continuous dark in comparison with the 12 h light/12 h dark regime (TABLE III). While on PDA the colony area yielding pycnidia and pycnidia density (number of pycnidia in a 1 cm diam field) were affected by light regimes, but the index of pycnidial production and number of conidia produced per plate was not significantly different between 12 h light/12 h dark and 24 h light regimes. On AJA, MEA and PJA, continuous light did not significantly affect pycnidial and conidial production in comparison with the 12 h light/12 dark regime.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and S. tumefaciens (Rodriguez et al 1985). On PDA at 20 C S. pyriputrescens colonies initially appeared colorless but started to turn light yellow to yellow after 5–7 d of incubation. This yellow pigmentation could be a useful characteristic in the diagnosis of Sphaeropsis rot caused by the fungus.
Results of temperature studies were in agreement with that of Xiao and Rogers (2004), but they tested only isolates from pears and did not test mycelial growth response at temperatures below 0 C. In the present study, six isolates of S. pyriputrescens recovered from different geographical regions in Washington and hosts were included and mycelial growth at –3 C also was tested. Rodriguez et al (1985) reported that the optimum temperature for mycelial growth of S. tumefaciens is 30–35 C. The optimum temperature for mycelial growth of S. sapinea is 25 C (Swart et al 1991, Jacobs and Rehner 1998). Lower temperature favored mycelial growth of S. pyriputrescens. An important characteristic for a fungus that is a postharvest pathogen on apple and pear is its ability to grow at fruit storage temperatures. Apples are commercially stored at –1 to 4 C and pears at –1.1 to 0.5 C (Meheriuk 1993). This study showed that S. pyriputrescens grows at –3 C, which indicates that Sphaeropsis rot caused by this fungus cannot be prevented by the low temperatures used for storage of apples and pears. In contrast, black rot caused by S. malorum Berk., a fruit-rotting pathogen in apples and pears, can be prevented by storage temperatures near 0 C (Snowdon 1992).
Increased radial growth of mycelium when water potentials decreased to approximately –2.0 MPa has been reported in other fungi such as Botrytis squamosa (Alderman and Lacy 1984), Fusarium sp. (Brownell and Schneider 1985, Cook and Duniway 1981), Gaeumannomyces graminis tritici (Cook and Duniway 1981), Macrophomina phaseolina (Olaya and Abawi 1996), Phytophthora sp. (Sommers et al 1970), Sclerotinia sclerotiorum (Grogan and Abawi 1975) and Verticillium dahliae (Ioannou et al 1977). The amount of stimulation and optimum water potential for mycelial growth depends on the fungus and, in some cases, temperature or other factors in the environment (Cook 1981). Most wood-destroying fungi are unable to grow at water potentials below –4 MPa (Carlile et al 2001). In the present study we found that the fungus grows at water potential as low as –5.6 MPa on KCl-amended PDA, indicating that S. pyriputrescens is a drought-tolerant fungal pathogen. Ability to invade water-deprived host tissues has been reported in other Sphaeropsis species. For example, increased disease severity was reported on water-deprived cypress (–4.5 to –5.5 MPa) caused by S. sapinea f. sp. cupressi (Madar et al 1989) and on water-deprived radiata and red pine trees (–1.9 to –2.5 MPa) caused by S. sapinea (Chou 1987, Blodgett et al 1997). S. pyriputrescens also has been found to be associated with a dieback and canker disease of crabapple trees in commercial apple orchards, and tree inoculation studies indicate that the fungus is able to cause cankers on apple and pear trees (Xiao et al unpubl). The ability of this fungus to grow at water potentials below –4 MPa might contribute partially to its adaptation to the dry climate of eastern Washington. The osmotic water potential ranges from –2.1 to –2.7 MPa in healthy stems and from –3.1 to –7.4 MPa in blackened stems (infected by fungi) of d’Anjou pear fruit (Sitton 1984). The water potentials of pear fruit stems are within the range of water potentials at which S. pyriputrescens is able to grow actively. The ability of the fungus to grow at the temperatures used for storage of pome fruits and at low water potentials might explain why the fungus is able to ramify the stem tissue of pear fruit to reach fruit flesh and cause fruit decay during storage (Xiao and Rogers 2004).
The pH range for active mycelial growth of S. pyriputrescens (3–6) and optimum pH (3–4) is similar to these reported for other fungi. The optimum pH for mycelial growth of most fungi is 5–6.5 (Ingold 1973). Rodriguez et al (1985) reported that optimum growth of S. tumefaciens is pH 4. The pH of fresh apple juice is 3.8 and pear juice 3.9, which are within the optimum pH for mycelial growth of S. pyriputrescens.
Lack of production of pycnidia and conidia in the dark and enhancement of pycnidial production by fluorescent light are in general agreement with reports for some fungi in Sphaeropsidales (Ekundayo and Haskins 1969, Kaiser 1973, Nebane and Ekpo 1992, McQuilken et al 1997). It generally is believed that lengthening light exposure increases pycnidial production. However, our result showed that the effect of light on pycnidial production was medium dependent (TABLE III). Pycnidial and conidial production on OMA was reduced under continuous light in comparison with the 12 h light/12 h dark regime. On AJA, MEA and PJA continuous light did not reduce pycnidial and conidial production compared with 12 h light/12 h dark. Nutrients and their availability on these media might be related to the differences in sporulation of the fungus in response to light regime.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: clxiao{at}wsu.edu
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LITERATURE CITED |
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———, Rogers JD, Boal RJ. 2004. First report of a new post-harvest fruit rot on apple caused by Sphaeropsis pyriputrescens. Plant Dis 88:223.
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