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© 2010 Plant Management Network.
Accepted for publication 26 July 2010. Published 27 August 2010.


Survey of Fusarium spp. Associated with Fruit Rot of Pumpkin in Ohio


Christian A. Wyenandt, former Graduate Research Assistant, Richard M. Riedel, Professor (retired), Landon H. Rhodes, Associate Professor (retired), Department of Plant Pathology, The Ohio State University, Columbus, OH 43210; Mark A. Bennett, Professor, Department of Horticulture and Crop Science, The Ohio State University, Columbus, OH 43210; and Steven G. P. Nameth, Director and Professor, Agricultural Technical Institute, The Ohio State University, Wooster, OH 44691


Corresponding author: Christian A. Wyenandt. wyenandt@aesop.rutgers.edu


Wyenandt, C. A., Riedel, R. M., Rhodes, L. H., Bennett, M. A., and Nameth, S. G. P. 2010. Survey of Fusarium spp. associated with fruit rot of pumpkin (Cucurbita pepo) in Ohio. Online. Plant Health Progress doi:10.1094/PHP-2010-0827-01-RS.


Abstract

From 2000 to 2002 commercial pumpkin (Cucurbita pepo) fields in Ohio were surveyed for Fusarium fruit rot. From six counties in 2000, 2001, and 2002, a total of 43 isolates from eight farms, 84 isolates from nine farms, and 40 isolates from six farms were collected, respectively. Fusarium solani f. sp. cucurbitae race 1 was the most commonly isolated Fusarium species from infected pumpkin fruit in Ohio. Other Fusaria isolated from infected fruit included F. oxysporum, F. graminearum, and F. acuminatum. A survey of infected pumpkin fruit in the field and inoculation studies of mature, healthy pumpkin cultivar Magic Lantern in the laboratory resulted in three types of fruit rot symptoms. Type 1, caused by F. oxysporum and F. acuminatum, resulted in a slow-expanding rot just below the rind surface of the infected fruit. Type 2, caused by F. graminearum, resulted in an expanding, slightly sunken, irregular rot of the rind surface. Type 3, caused by F. solani resulted in expanding, circular sunken lesions on the fruit surface bearing white to tan sporodochia. This is the first report of F. solani f. sp. cucurbitae race 1, F. oxysporum, F. acuminatum, and F. graminearum causing fruit rot of pumpkin in Ohio.


Introduction

Pumpkins are an important cash crop in Ohio and are grown by wholesalers who sell by the tonnage or by retailers who operate roadside farm-markets. Most pumpkins grown in the state are sold for decorative purposes for fall holidays. Growers who operate roadside markets often plant pumpkins in “u pick” fields near the farm market as a way to attract customers during the fall season. As a result, pumpkins are planted in the same field on some of these farms each year. Thus, crop rotations of three or more years to help reduce soil-borne diseases are not practiced on some small roadside farms operations in Ohio, and yield loss due to diseases such as Fusarium fruit rot can be as high as 100% in some years (Mac Riedel, personal communication).

Fusarium fruit rot, caused by Fusarium spp., is an important soil-borne fungal disease of pumpkin in Ohio. At least 10 species of Fusarium are known to cause fruit rot in cucurbits in the United States, including F. equiseti, F. acuminatum, F. graminearum, F. avenaceum, and F. solani (1,3,9). In 1961, Toussoun and Snyder identified two races of F. solani f. sp. cucurbitae. F. solani f. sp. cucurbitae race 1 was determined to cause a root, seedling, and fruit rot, where race 2 caused only a fruit rot in cucurbit crops (9). Interestingly, F. solani f. sp. cucurbitae race 2 was collected only in California and Ohio at the time of its original designation in 1961. Since that time it has been suggested that F. solani f. sp. cucurbitae race 1 and 2 are related but distinct species (8). To date, Fusaria which cause fruit rot in pumpkin have only been identified to the genus level in Ohio (2).

In 1996, Elmer categorized symptoms of fruit rot caused by Fusarium species in Connecticut. Type 1, was described as a pre-harvest, dry hard rot and type 2 was described as a post-harvest, soft, sunken rot. According to Elmer (4) different species of Fusarium could cause either a type 1 or type 2 fruit rot, or both. The type 1 fruit rot was commonly observed on immature green fruit in the field (Fig. 1) and type 2 was often associated on fruit after harvest (Fig. 2).


 

Fig. 1. Symptoms (Type 1) of Fusarium fruit rot of immature green pumpkin fruit of pumpkin cultivar Magic Lantern caused by F. solani f. sp. cucurbitae race 1.

 

Fig. 2. Symptoms (Type 2) of Fusarium fruit rot of mature orange pumpkin fruit of pumpkin cultivar Magic Lantern caused by F. solani f. sp. cucurbitae race 1.


Objectives of this study were: (i) to survey commercial pumpkin fields in Ohio for the presence of Fusarium fruit rot; (ii) to isolate and identify common Fusarium species causing or associated with fruit rot of pumpkin; and (iii) to determine if symptoms of Fusarium fruit rot on mature pumpkin can be associated with specific Fusarium spp. A bioassay was conducted to determine if infested seed associated with fruit rot could cause a seedling rot in germinating seed.


Isolation of and Determination of Fusarium spp.

In 2000, 2001, and 2002 commercial vegetable farms in central and northern Ohio which produce pumpkins for retail (i.e., road-side markets, u-pick operations) or wholesale were surveyed for Fusarium fruit rot development. In 2000, 2001, and 2002, respectively, 43 isolates from eight farms, 84 isolates from nine farms, and 40 isolates from six farms were collected (Table 1). Prior to isolation, each pumpkin fruit was visually examined. All lesions on infected fruit from which isolations were made were labeled and digitally photographed and cataloged for later comparisons. All fruit were surface sterilized by rinsing the infected area with 95% ethyl alcohol prior to isolations. Areas of rind from the margins of lesions were removed, and infected tissue was excised and placed on 100 × 15-ml petri plates (Fisher Scientific Co., LLC Agawam, MA) containing potato dextrose agar (PDA) and/or acidified potato dextrose agar (APDA) and incubated at 25°C under 12-h light and dark intervals for identification purposes (2,6). Hyphal tip transfers from each isolate to new PDA culture plates or APDA in slants were done for longer term storage (6 to 8 weeks) in an incubator at 25°C under 12-h light and dark intervals. Additionally, hyphal tip transfers were placed onto carnation leaf agar (CLA) according to Burgess et al. (2) to allow for sporodochia (i.e., macroconidia) development. After three to four weeks isolates were examined for phenotype (i.e., colony type and color) and morphological characteristics such as sporodochium development, phialide type, macro- and microconidia and/or chlamydospore development. Based on these characteristics isolates could be identified to species level based on descriptions by Burgess et al. (2) and Nelson et al. (5).


Table 1. Fusarium species identified by farm and county in Ohio from
2000 to 2002.

Year Farm County Fusarium spp. isolated*
2000 A Franklin F. solani, F. oxysporum
B Champaign F. solani, F. acuminatum
C Licking F. solani
D Holmes F. oxysporum
E Franklin F. oxysporum
F Wayne F. solani
G Sandusky F. solani
H Wayne F. graminearum
2001 A Licking F. solani
B Pickaway F. solani
C Pickaway F. solani
D Franklin F. solani
E Wayne F. graminearum
F Ottawa F. solani
G Wayne F. solani
H Pickaway F. solani
I Holmes F. solani
2002 A Sandusky F. solani, F. oxysporum
B Pickaway F. solani
C Franklin F. oxysporum
D Wayne F. solani, F. oxysporum
E Licking F. solani
F Pickaway F. solani

 * Number of isolates from each farm not shown.


Classification of External and Internal Fusarium Fruit Rot Symptoms on Mature Pumpkin Fruit

Healthy fruit of pumpkin cultivar Magic Lantern (Harris Moran Seed Company, Modesto, CA) were inoculated with isolates collected during each year of the study to determine (i) proof of pathogenicity and (ii) if different Fusarium spp. could cause distinguishable external and internal symptoms on infected fruit. In each year mature, healthy ‘Magic Lantern’ fruit were inoculated using a cut inoculation technique. Three- to four-week-old Fusarium isolates grown on PDA or APDA medium were used to inoculate healthy pumpkin cultivar Magic Lantern fruit by removing a 1-cm² area of pumpkin fruit tissue and replacing it with a 1-cm² plug of agar with active mycelia. Excised pumpkin tissue was returned to the site of inoculation and a piece of clear tape (i.e., Scotch) was used to hold the tissue in place. A mature, individual fruit was inoculated 4 to 5 times with the same isolate for replication to determine if similar symptoms developed from each inoculation site. As a control, a 1-cm² area of pumpkin fruit tissue was excised from each fruit and immediately replaced and a piece of clear tape was used to hold tissue in place. All inoculated pumpkin fruit were stored in a laboratory at room temperature (18 to 21°C) for up to ten weeks after inoculation to allow for symptom development. Re-isolations from all replicated inoculations were conducted using the same techniques described above to complete Koch’s postulates. External symptoms were monitored for up to ten weeks after each inoculation to allow for lesions to develop. During this time, each inoculated fruit was compared to photos of the original symptoms that were present on the infected fruit during the initial isolation process. After visual examinations, each fruit was cut open and internal infection characteristics were described.


Infested Seed and Seedling Bioassays

Three- to four-week-old cultures of selected Fusarium spp. isolates from each farm were used to inoculate individual, healthy mature fruit of pumpkin cultivar Magic Lantern using the cut inoculation technique described above. Not all isolates from each farm were used since the same species of Fusarium (i.e., Fusarium solani) was isolated multiple times from different fruit from the same farm each year. All inoculated pumpkin fruit were incubated in a dry laboratory environment (18 to 21°C) for up to ten weeks after inoculation. After each inoculated fruit showed visible symptoms of Fusarium fruit rot (greater than 50% total rot) or reached 10 weeks after inoculation, all seeds from infected fruit were removed, dried, and stored for future use in seedling bioassays.

In each year, clean (non-infested) and Fusarium-infested seed were used in a seedling bioassay to determine if seed collected from inoculated fruit could cause a seedling rot. Pumpkin seed infested with Fusarium typically have a distinctive bluish-green or darkened color to their seed coat and can easily be separated from non-infested seed (although some seed without discoloration may still be infested). Sterilized plastic pots (4 inches in diameter) were filled with potting soil (Metro-Mix 360, Scotts-Sierra Horticultural Supply, Marysville, OH) that had been previously autoclaved for two hours at one-hour intervals. Five infested seeds with discoloration due to Fusarium infestation collected from each inoculated fruit were seeded in a pot (4 replications each, 20 seeds total) and placed under an intermittent mist system in a greenhouse with normal daylight hours and a temperature between 18 and 21°C. Additionally, five clean seed were seeded in a pot as a control. Seedlings were rated at one and three weeks after sowing to determine if isolates of F. solani, F. oxysporum, F. graminearum, or F. acuminatum could cause a seedling rot. At one and three weeks after sowing, seedlings were rated as positive (+) with symptoms of Fusarium fruit rot (FFR) development or negative (-) with no visible FFR symptoms. Additionally, seedlings were rated and described according to origin of rot (i.e., infection starting at the base of the hypocotyl or infection starting at the base of cotyledons).


Survey Results and Symptom Classification

In this study, infected pumpkin fruit were collected to determine which Fusarium spp. were most prevalent in commercial fields in Ohio from 2000 to 2002. Phenotypic and morphological features such as colony color, phialide type, micro- and macroconidia development, seedling bioassays, and proof of pathogenicity tests as described by Summerell et al. (7) and others (2,3,6) were used to characterize and determine which Fusarium species were associated with causing fruit rot of pumpkin (C. pepo). F. solani f. sp. cucurbitae was isolated from symptomatic fruit from 18 of 23 farms (78%) surveyed during the course of this study (Table 1). Other species identified included F. oxysporum, F. graminearum, and F. acuminatum. Fusarium oxysporum was isolated from infected fruit collected from five farms, F. graminearum was isolated from infected fruit collected from two farms, and F. acuminatum was isolated once from an infected fruit collected during the 2000 production season (Table 1). On some farms, more than one Fusarium species were isolated from infected pumpkin fruit. However, during the three years of this study F. solani f. sp. cucurbitae was the most predominant species associated with causing fruit rot of pumpkin in Ohio (Table 1).

The symptoms that were present on infected fruit collected from commercial fields and developed on inoculated fruit in the laboratory were categorized into three types of fruit rot. Type 1, caused by F. oxysporum and F. acuminatum, was characterized as a dry, slow expanding rot of mature pumpkin fruit tissue just below the rind surface (Fig. 3). Over time (in most cases, more than four weeks) this type of rot could extend into the seed cavity and be accompanied by white mycelial growth typical of F. oxysporum (Fig. 4) or light pink-colored mycelial growth typical of F. acuminatum in culture (Fig. 5). Type 2 fruit lesions, caused by F. graminearum, were categorized as an expanding, irregular external and internal rot with red-colored mycelial growth in the pumpkin seed cavity (Fig. 6). External symptoms on mature infected fruit characteristic of F. graminearum were characterized by a slightly sunken, irregular rot of the rind surface (Fig. 7). Type 3 fruit lesions, characteristic of F. solani f. sp. cucurbitae, were categorized as an expanding internal rot with characteristic whitish-green mycelial growth and a greenish-brown discoloration of the seed (Fig. 8). External symptoms on mature infected fruit were characterized by slightly sunken, expanding lesions bearing numerous white to tan sporodochia in centers (Fig. 9).


 

Fig. 3. Symptoms of internal fruit rot caused by artificial inoculation of mature orange of pumpkin cultivar Magic Lantern with F. oxysporum.

 

Fig. 4. Symptoms of internal seed cavity rot of pumpkin cultivar Magic Lantern caused by artificial inoculation with F. oxysporum.


 

Fig. 5. Symptoms of internal seed cavity rot of pumpkin cultivar Magic Lantern caused by artificial inoculation with F. acuminatum.

 

Fig. 6. Symptoms of internal seed cavity rot of pumpkin cultivar Magic Lantern caused by artificial inoculation with F. graminearum.


 

Fig. 7. Symptoms of Fusarium fruit rot of pumpkin cultivar Magic Lantern artificially inoculated with F. graminearum.

 

Fig. 8. Symptoms of internal seed cavity rot of pumpkin cultivar Magic Lantern caused by artificial inoculation with F. solani.


 

Fig. 9. Typical symptoms of Fusarium fruit rot of pumpkin cultivar Magic Lantern caused by F. solani f. sp cucurbitae race 1.

 

The symptoms caused by F. solani f. sp. cucurbitae race 1 fruit rot seen in the field and in proof of pathogenicity tests on cv. Magic Lantern fruit in the laboratory are in accordance with Elmers’ (4) description of two types of fruit rot developing in the field. Typical symptoms of fruit rot caused by Fusarium spp. in the field often were seen as a non-expanding dry rot of immature pumpkin fruit (Type 1) or a slightly-sunken, soft expanding rot of mature fruit (Type 2). Although Elmer described type 1 as being a pre-harvest rot and type 2 as being a post-harvest rot, F. solani f. sp. cucurbitae isolated from infected fruit in the field during the production season in Ohio in this study caused a pre-harvest soft, sunken, expanding rot and was classified as Type 3 rot.

Elmer (4) stated that it was possible for Type 1 lesions (rots) to develop into Type 2 lesions (rots). This was observed when symptoms on immature (green) fruit of cultivar Magic Lantern infected with F. solani f. sp. cucurbitae progressed from a dry non-expanding rot to a soft, slightly sunken expanding rot as the fruit matured in the field (Figs. 1 and 2). In this study, Type 1 lesions that developed on inoculated pumpkin cultivar Magic Lantern fruit in laboratory tests were very similar to symptoms present in the field, and were similar to descriptions by Mehl et al. (8). Bruton and Dunthie (2) cautioned that the symptoms expression on fruit of cucurbits could be strongly influenced by time of infection and environmental conditions. In accordance with Elmer (4), it could be said that the Type 3 rot seen in this study could be classified as a pre-harvest rot characterized by the similar symptoms, and referred to as a post-harvest (Type 2) rot in Elmer’s classification scheme and later described by Mehl et al. (8).

In this study, two types of seedling rot caused by F. solani f. sp. cucurbitae race 1 were categorized (data not shown). Type 1 occurred as cotyledons emerged from the infested seed coat during germination resulting in an infection at the base of the cotyledon leaf (Fig. 10). These seedlings often rotted at the soil line as the cotyledons emerged from the soil surface, and in some instances sporodochia developed on infected tissue. Type 1 seedling rot was most predominant during germination of Fusarium solani infested seed in sterilized potting soil (Fig. 11). In other instances, seedlings germinated and grew with lesions developing from the growing tip of the seedling toward the base of the hypocotyls (Fig. 12). These lesions (classified as Type 2 lesions), were most noticeable when clean, non-infested seed was germinated in a Fusarium solani-infested potting soil. Type 2 seedling rot resulted in white to tan lesions developing on infected hypocotyls (Fig. 13) (data not shown). Microscopic examination of infected hypocotyl tissue showed intra- and intercellular colonization of tissue by F. solani f. sp. cucurbitae race 1. F. graminearum, F. acuminatum, or F. oxysporum infested seeds germinated without any type of symptom development, and clean seed sown in infested potting soil containing each pathogen also germinated without any associated symptoms (data not shown). Interestingly, races of F. oxysporum exist and can cause a damping-off and wilt in other cucurbits, such as watermelon caused by F. oxysporum f. sp. niveum and melon caused by F. oxysporum f. sp. melonis. No such race of F. oxysporum exists that can cause a damping-off or wilt in pumpkin (C. pepo). In proof of pathogenicity tests, F. oxysporum was able to cause a limited fruit rot. Although this type of rot was slow developing, in some instances the fungus was able to penetrate the seed cavity when inoculated fruit were allowed to sit in a dry environment for extended periods of time (i.e., up to 10 weeks after inoculation). The fruit rot caused by F. oxysporum seen in this study was most likely a result of the inoculation technique. This is in accordance with Elmer (4) who stated that in some instances when F. oxysporum was inoculated onto pumpkin cultivar Howden the pathogen was able to colonize the perimeter of the wound as the fruit began to age and collapse. It is most likely that the F. oxysporum isolates obtained during this study were opportunistic pathogens and only able to colonize fruit after some other pathogen infected the fruit.


 

Fig. 10. Infection at base of a cotyledon leaves causing type 1 seedling rot by F. solani f. sp. cucurbitae race 1 from an infested seed grown in sterilized potting soil.

 

Fig. 11. Newly emerged seedling of pumpkin cultivar Magic Lantern infected by F. solani f. sp. cucurbitae race 1 at base of cotyledon during germination.


 

Fig. 12. Symptoms of seedling rot (Type 1) in pumpkin cultivar Magic Lantern caused by F. solani f. sp. cucurbitae race 1 from an infested seed germinated in sterilized potting soil.

 

Fig. 13. Seedling rot (Type 2) in pumpkin cultivar Magic Lantern with developing aerial mycelium caused by F. solani f. sp. cucurbitae race 1 from artificially-inoculated potting soil.


The importance of macro- and microconidia production in overwintering of Fusarium spp. in pumpkin (C. pepo) is not well understood since it is possible for mycelium to overwinter inside or on the seed coat. In some instances, chlamydospores were seen to develop in heavily infected fruit tissue (F. solani and F. oxysporum), but this was not evident on macroscopic examination of numerous inoculated fruit that were kept in a dry, warm environment. Studies have shown that approximately 30% to 50% of the macroconidia produced from sporodochia or conidia on the stem near the ground surface either deposited in the soil by moisture or by mechanical means are converted to chlamydospores in one to eight weeks, and most of these chlamydospores do not survive for more than one year (4). Although chamydospores are known to play key roles as survival structures in other formae speciales of Fusarium solani, their exact role in the etiology of Fusarium fruit rot development in pumpkin remains unknown.


Conclusions and Recommendations

Eighty to one hundred percent of the pumpkin fields surveyed in Ohio from 2000 to 2002 had some incidence of Fusarium fruit rot. During the course of this study, F. solani f. sp. cucurbitae race 1 was the most commonly identified Fusarium species associated with fruit rot of pumpkin in Ohio. F. solani f. sp. cucurbitae race 2 was not found during this study, although it had been identified in Ohio in 1961 (9).

Importantly, reasons why the seedling rot phase of F. solani f. sp. cucurbitae is not commonly found in Ohio pumpkin fields infested with the pathogen include: (i) this phase of the disease may go unnoticed because other soil-borne fungi may also cause a seedling rot; (ii) inoculum levels of the pathogen in the field may not be high enough to cause rot in enough seedlings to be noticed by a farmer; (iii) pumpkins may be seeded at a time when environmental factors do not favor disease development or support population growth of the pathogen in the soil; or (iv) under field conditions, pumpkin plants may be able to tolerate a low rate of root infection by producing enough new root growth to outgrow the damage done by the pathogen.

The most distinguishing characteristic of F. solani f. sp. cucurbitae race 1 is the fruit rot stage of the disease. It is known that infested seed from infected fruit left in pumpkin fields will germinate during the following spring and summer months. Because infected pumpkins may be an important source of inoculum, removing fruit infected with Fusarium fruit rot from the field may be a useful practice, especially if pumpkins are planted continually in the same field or under short (1 or 2 year) rotations.


Literature Cited

1. Bruton, B. D., and Duthie, J. A. 1996. Fusarium fruit rot. Pages 50-51. In: Compendium of Cucurbit Diseases. T. A. Zitter, D. L. Hopkins, and C. E. Thomas, eds. American Phytopathological Society, St. Paul, MN.

2. Burgess, L. W., Summerell, B. A., Bullock, S., Gott, K. P., and Backhouse, L. W. 1994. Laboratory Manual for Fusarium Research, 3rd Edn. University of Sydney/Royal Botanic Gardens, Sydney, Australia.

3. Ellett, C. W. 1989. Ohio Plant Disease Index. Special Circ. 128. Ohio Agric. Res. and Development Center, The Ohio State University, Wooster, OH.

4. Elmer, W. H. 1996. Fusarium fruit rot of pumpkin in Connecticut. Plant Dis. 80:131-135.

5. Nelson, P. E., Toussoun, T. A., and Marasas, W. F. 1983. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State Univ. Press, University Park, PA.

6. Nash, S. M., and Alexander, J. V. 1965. Comparative survival of F. solani f. cucurbitae and F. solani f. phaseoli in soil. Phytopathology 55:963-966.

7. Summerell, B. A., Buharuddin, S., and Leslie, J. F. 2003. A utilitarian approach to Fusarium identification. Plant Dis. 87:117-128.

8. Mehl, H. L., and Epstein, L. 2007. Identification of Fusarium solani f. sp. cucurbitae Race 1 and 2 with PCR and production of disease-free pumpkin seed. Plant Dis. 91:1288-1292.

9. Toussoun, T. A., and Snyder, W. C. 1961. The pathogenicity, distribution, and control of two races of Fusarium (Hypomyces) solani f. cucurbita. Phytopathology 51:17-22.