Mycologia
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DOI: 10.3852/mycologia.97.4.901
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Mycologia, 97(4), 2005, pp. 901-907.
© 2005 by The Mycological Society of America

Survival, growth and pathogenicity of Gaeumannomyces graminis var. graminis with different methods of long-term storage


M.L. Elliott 1

     University of Florida, Fort Lauderdale Research and Education Center, Department of Plant Pathology, 3205 College Avenue, Fort Lauderdale, Florida 33314

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

The fungal plant pathogen Gaeumannomyces graminis var. graminis was preserved with 12 different storage methods. Five strains, each with unique morphological and pathological characteristics, were used for comparison of the methods. The storage treatments included potato-dextrose agar slants, with or without mineral oil, stored at either 4 C, 28 C or ambient temperature; colonized agar plugs placed in glycerol solution at either –75 C or –20 C; colonized agar plugs placed in sterile deionized water at either 4 C or ambient temperature; and mycelial growth on intact or precut pieces of filter paper, desiccated and stored at ambient temperature. Survival was evaluated at 6, 12, 24, 36, 48 and 120 mo. The three best treatments for survival were PDA slants, with or without mineral oil, and colonized agar plugs stored in water, all at ambient temperature. All five fungal strains were recovered from all four replicates at each sampling date for agar plugs stored in water at ambient temperature. The worst treatments were agar slants and agar plugs in water stored at 4 C and agar plugs stored in glycerol at –20 C. Morphological characteristics were not affected by storage treatments. In general, there were minimal or no effects on growth and pathogenicity for all strains for all storage treatments with survival. Colonized agar plugs stored in water at ambient temperature provides an economical storage method (materials and labor) that does not need an electrical power for long-term maintenance.

Key words: culture maintenance, culture storage, preservation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
One of the most difficult aspects of long-term research on a specific host-pathogen system is maintenance of isolates. The same isolate(s) or strain(s) is(are) ideally used each time to culture fungal pathogens so that meaningful comparisons can be made between experiments, which may be conducted during several years. This is especially true when evaluating host germ plasm. Maintenance of well characterized isolates is also important for genetic studies of the pathogen and for development of diagnostic assays with national or international value. Fungal preservation methods relative to fungal survival have been examined in numerous studies (e.g. Boesewinkel 1976Go, de Capriles et al 1989Go, Carter and English 1994Go; Sharma and Smith 1999Go). Some researchers (e.g. Burdsall and Dorworth 1994Go) have gone further to examine growth rate of the fungal strains that survived, and a few (Butler 1980Go; Windels et al 1993Go, Hajek et al 1995Go) have examined the effect of storage on plant pathogenicity and virulence, especially for long-term storage.

Numerous methods are available for storage, with lyophilization or cryopreservation in liquid nitrogen or a mechanical deep freeze at –70 C most often cited as the preferred methods for maintenance (Ryan et al 2000Go; Smith and Onions, 1994Go). All three methods require special equipment; the mechanical deep freeze uses electricity, and liquid nitrogen must be replenished routinely. More economical plant pathogen preser vation techniques include agar slants, often under oil to prevent dehydration, water, silica gels and even dried soil (Tuite 1969Go, Smith and Onions 1994Go). The temperature used in these storage methods is variable, from –20 C to ambient temperature.

This study was initiated to evaluate storage methods for Gaeumannomyces graminis (Sacc.) Arx & D. Olivier var. graminis, a pathogen of Poaceae species grown in tropical, subtropical and southern temperate climates (e.g. Elliott 1991Go, Webster and Gunnell 1992Go, Wilkinson 1994Go, Wong et al 2003Go). Other varieties of G. graminis include avenae, maydis and tritici, all primarily pathogens of Poaceae species (Walker 1981Go, Hornby et al 1998Go). Studies with G. graminis pathogens have demonstrated that loss of pathogenicity is a potential problem when the fungus is stored on agar or repeatedly subcultured, as is often the case when stored on agar (Hornby et al 1998Go, Elliott 1995Go). One study demonstrated that pathogenicity was lost when the agar slant was not covered with oil before storage at 5 C (Bunny 1981Go). Other reports indicate that storage in liquid nitrogen maintains pathogenicity but that storage in water does not (Hornby et al 1998Go). The primary goal of this study was to determine if G. graminis could be stored at ambient temperature, thus eliminating dependence on special equipment or electricity. If maintenance of a specific temperature was required, then the next goal was to determine which temperature and storage method were best for survival of the pathogen. In both cases the interest was not just in survival of the fungal isolate but also maintenance of its morphological and pathological characteristics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Fungal isolate and strains.— – Five strains of G. g. graminis were selected, each with a unique combination of morphological and pathological characteristics. The original isolate of G. g. graminis was obtained from diseased St Augustinegrass (Stenotaphrum secundatum) roots in 1988 and designated FL-39. It had been characterized (Elliott 1991Go, Elliott and Landschoot 1991Go). Strains of FL-39 were obtained by protoplasting hyphae that, in some experiments, were subjected to chemical mutagenesis with N-methyl-N’-nitro-N-nitrosoquanidine (MNNG) (Sigma, St Louis, Missouri) before protoplasting (Elliott and Chasse 1998Go), a technique that yields variation in morphological and pathological characteristics of fungi (Boland and Smith 1991Go, Yang et al 1994Go). Characteristics of the original isolate and four strains used in this study are provided (TABLE IGo). The strains can be distinguished easily from one another and the parent isolate.


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  		TABLE I. Characteristics of Gaeumannomyces graminis var. graminis isolate FL-39 and strains used for assessment of storage methods, with strains obtained by chemical mutagenesis and protoplasting hyphae of FL-39

 
Storage methods.— – Twelve storage treatments were evaluated. Details of each treatment are provided below and summarized (TABLE IIGo). All materials were obtained from Fisher Scientific (Pittsburgh, Pennsylvania) or Sigma. Fungal strains were grown 5 d on potato-dextrose agar (PDA) (Difco) to study treatments using colonized agar plug. Plugs were cut with a sterile 5 mm cork borer. In the case of six storage treatments, the storage unit was a 4 mL clear glass sample vial sealed with a rubber-lined screw cap (Kimble). Each vial was filled with 1.5 mL of PDA, sterilized and solidified at a slant. Each vial was inoculated with one colonized agar plug, with 30 inoculated vials per strain, incubated 2 wk at 28 C, then separated into the six storage treatments. Only those vials with normal growth covering the agar slant were selected. In the case of three treatments, sterile mineral oil (Sigma, molecular biology grade) was added to the vial until the agar was covered. Eight vials per strain (four with and four without mineral oil) were stored at 4 C, 28 C and ambient temperature. Normal ambient temperature was 24 C, but the temperature was highly variable throughout the experiment with high temperatures reaching 32 C or higher for more than 24 h due to loss of air conditioning in the building. All vials were sealed with Parafilm® prior to storage in the dark.


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  		TABLE II. Summary of methods used for storage of Gaeumannomyces graminis var. graminis strains

 
Four storage treatments used sterile cryogenic 2.0 mL polypropylene, external thread vials with screw caps (Corning) as the storage unit. In the case of two treatments, the vials contained 1.5 mL of a 40% glycerol solution. Eight colonized agar plugs were placed in each glycerol-containing vial, minimizing the amount of agar used. In the case of each fungal strain, four of these vials were placed at –75 C and four were placed at –20 C. The vials contained 1.5 mL sterile deionized water for two treatments. Again, eight colonized plugs were placed in each water-containing vial, minimizing the amount of agar used; vials were sealed with Parafilm®. Four of these vials were stored at 4 C and four were stored at ambient temperature for each fungal strain. All vials were stored in the dark.

Two storage treatments consisted of fungal mycelium grown across a sterile filter paper (7 cm diam, Whatman No. 4) placed on PDA in a Petri plate. After 2 wk at 28 C, the filter paper was removed from the agar plate and dried in a desiccator with DrieriteTM- absorbent. There were two colonized filter papers per strain. A sterile blade was used to cut each filter paper into approximately 3 mm square pieces. These pieces were divided into four portions, and each portion placed in a sterile 4 mL glass vial as used for the PDA slants. The vials were sealed with Parafilm® and stored at ambient temperature in the dark. The second filter paper was left whole and placed in a heat-sealed bag (Dazey Seal-a-Meal; 2 mil thick) that was stored at ambient temperature in the dark.

Determination of storage survival.— – At specific times after the initial storage date of Dec 1994, strains were transferred from each replicate of each storage treatment to PDA to determine survival of the fungal strain. Agar plugs retrieved from glycerol solution or water first were blotted dry on sterile filter paper. In the case of the whole filter paper treatment, four pieces were cut from the paper with a sterile blade. Plates were examined daily for 10 d. If there was no growth of any of the four replicates of a storage treatment, the strain was transferred again to ensure that the strain was not viable in that storage treatment on that sampling date.

Survival assays were conducted at 6, 12, 18, 24, 36, 48 and 120 mo after the initial storage date. When no growth of a strain had been obtained from any replicate of a storage treatment for at least two consecutive sampling times, that treatment for that particular strain no longer was assayed for survival.

Linear growth on PDA.— – At the time the storage survival assays were conducted, the linear growth of each fungal strain x storage treatment was evaluated for comparison with the linear growth of the strain at the initial storage date. A colonized PDA plate of one replicate of each storage treatment for each strain was selected for growth rate evaluation. Plugs (5 mm diam) were transferred to the center of fresh PDA plates. There were four plates (replicates) for each fungal strain x storage treatment. Radial growth was measured after 4 d at 28 C.

Water agar/wheat seedling assay for pathogenicity.— – At the time storage survival was determined a pathogenicity assay also was conducted for comparison with pathogenicity of the strain at the time of the initial storage date. Pathogenicity was assayed using an in vitro method wherein wheat seeds were planted on water agar followed by inoculation with the fungal strain (Speakman 1982Go, Elliott and Landschoot 1991Go). This assay also allows for observation of hyphopodia and perithecial production. Surface-sterilized wheat seeds, three per plate, were germinated on 1.5% water agar. For each strain, a 5 mm diam fungal plug, obtained from PDA colonized with each strain x storage treatment, was placed next to each germinated seed. Nine seedlings were inoculated for each strain x storage treatment. The control treatment was uncolonized PDA plugs placed next to seedlings. Plates were sealed with Parafilm® and incubated 21 d at 25 C with 12 h light/d.

Plants were evaluated for disease with this scale: 1 = plants healthy with white roots; 2 = plants healthy but roots discolored (tan not white); 3 = majority of roots black, basal stem white, <50% chlorotic leaves; 4 = all roots black, basal stem black, 50–75% chlorotic or necrotic leaves; 5 = roots and basal stem black, >75% chlorotic or necrotic leaves.

Statistical analysis.— – Analysis of variance was performed with SAS procedure GLM (SAS version 6.12, SAS Institute Inc., Cary, North Carolina). Using Dunnett’s t-test, means for linear growth and disease rating for each strain x storage method were compared against linear growth and disease rating for the strain at the initial storage date.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
In this study, the three best treatments for storage survivability were PDA slants stored at ambient temperature, with or without mineral oil, and colonized agar plugs stored in sterile deionized water at ambient temperature (TABLE IIIGo). All five fungal strains were recovered from at least one of four replicates of each of these treatments on each sampling date. Of the three, recovery from all four replicates of each strain for each sampling date was obtained only with the colonized agar plugs stored in water. It should be noted that after 120 mo many of the PDA slants without mineral oil had dried out, but all the strains had at least one viable replicate slant.


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  		TABLE III. Months of survival of five Gaeumannomyces graminis var. graminis strains using twelve different storage methods

 
In the cases of all strains except the parent isolate FL-39, PDA slants covered with mineral oil and stored at 28 C permitted recovery from at least one replicate on each sampling date (TABLE IIIGo). PDA slants without mineral oil and stored at 28 C permitted recovery from at least one replicate of each strain for at least 36 mo and for 120 mo for three of the strains. Storage on PDA slants, with or without mineral oil, or agar plugs in water at 4 C were three of the worst storage treatments. Other storage treatments with limited or no recovery of fungal strains were colonized agar plugs in glycerol at –20 C and the dried filter paper methods.

In general the strains maintained their individually distinct morphological colony characteristics (personal observation, see TABLE IGo). If FL-39 was recovered, it continued to produce perithecia. FL-39-21 continued to produce abnormal hyphopodia, whereas all other strains continued to produce normal hyphopodia. FL-39-106, FL-39-111 and FL-39-114 continued to produce their unique colony characteristics. Concerning the three best methods, the linear growth of all strains was not significantly affected by these treatments when compared across all sampling dates to the linear growth at the initial storage date (TABLE IVGo). Likewise, with one exception, the pathogenicity and virulence of the strains were not significantly affected by the three best treatments (TABLE IVGo). The exception was FL-39 when the PDA slant with mineral oil was stored at ambient temperature. In this case the virulence decreased.


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  		TABLE IV. Comparison of storage method and its effect on linear mycelial growth and disease rating for five Gaeumannomyces graminis var. graminis strains

 
FL-39-106 survived less well than the other strains (only 5 of 12 methods), but when recovered its morphological and pathological characteristics were not significantly altered from when it was placed in storage. Remaining strains were recovered from either six (FL-39), seven (FL-39-111), eight (FL-39-114) or 10 (FL-39-21) different storage methods. Each of these strains had at least one storage method that significantly altered either growth or pathogenicity. However, when compared to the initial storage date values for these characteristics, no one storage method consistently altered these characteristics.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Ryan et al (2000)Go have provided a decision-based key for determining storage methods best suited for fungal cultures. An examination of this key relative to G. graminis illustrates the difficulty in deciding which storage method to use for this fungus and similar fungi. Because G. graminis rarely produces asexual spores in culture, is not readily maintained by continual subculture and is not endomycorrhizal, the "species-specific criteria" portion of the key indicates that if the fungus does not produce sexual structures then one should "consider preservation with substrate from which the fungus was isolated." Many G. graminis isolates do not produce sexual structures. If the fungus does produce sexual structures, then preservation by cryopreservation or lyophilization is recommended. However these authors correctly point out that storage of sexual structures, perithecia with ascospores for G. graminis, may result in fungal characteristics that are different from the parent isolate.

The primary goal for examining long-term storage methods for G. g. graminis was to learn how to effectively and economically store this fungal pathogen so that morphological and pathological characteristics are maintained over long periods. This would permit long-term evaluations and comparison of studies conducted at different times by the same researcher or different researchers. While cryopreservation below –70 C is an effective storage method, isolate collections stored with this method require reliable electrical service, service which is vulnerable to loss in catastrophic events (e.g., hurricanes, floods, fires, etc.). Stored lyophilized cultures usually are refrigerated after processing and therefore are vulnerable to electrical service disruptions, but they can be stored at ambient temperatures. However specialized equipment and considerable labor is required in the initial storage process. Furthermore this technique is more applicable to fungi that produce asexual spores, a characteristic not associated with G. graminis.

Hornby et al (1998)Go noted that G. g. tritici and G. g. avenae collections stored in liquid nitrogen did retain pathogenicity up to 12 y. They also noted that isolates of G. graminis (the variety is not specified) stored as agar plugs in sterile water at ambient temperature have survived more than 20 y. However, with notable exceptions, pathogenicity was not expected to be maintained after more than 1 or 2 y. In the current study, water storage did not appear to affect pathogenicity or virulence on wheat of the five strains, even after 10 y.

As observed in other water storage studies, storage of the vials at ambient temperature was adequate for recovery (Boesewinkel 1976Go, Ellis 1979Go, Jones et al 1991Go). In fact in the present study refrigeration was detrimental in the recovery of G. graminis. This might be due to the semitropical origin of the G. g. graminis isolate. I emphasize that the ambient temperature in which these strains were maintained varied considerably. While the temperature goal in the building is 24 C, loss of air conditioning in the summer months in Florida causes the temperature to increase rapidly.

Recovery from the agar slant storage method, either with or without mineral oil covering the slant, also was unaffected by an inconsistent ambient temperature. Furthermore morphological and pathological characteristics were unaffected. This is in contrast to an observation by Bunny (1981)Go where a G. g. tritici strain recovered from an agar slant not protected by oil was less virulent on wheat than the same strain that was protected by oil. The use of oil is recommended because many of the agar slants without oil in the current study dehydrated after 10 y.

The most unexpected result from this study was lack of continued recovery from the agar plugs stored in glycerol solution at –75 C. This form of cryopreservation usually is considered to be reliable. However one strain (FL-39-106) in this study was never recovered under this storage method (i.e. it did not survive 6 mo). Therefore, even if a reliable electrical source was not a consideration, it still would not be the storage method of choice for this fungus.

In summary water storage at ambient temperature provides an economical method for long-term preservation of the plant pathogen G. g. graminis. It is a method that does not rely on electricity or special equipment for long-term maintenance. This storage method does not appear to affect the morphological or pathological characteristics of the fungus.


   ACKNOWLEDGMENTS
 
The author thanks E.A. DesJardin for technical support. This research was supported by the Florida Agricultural Experiment Station and approved for publication as Journal Series No. R-10742.


   FOOTNOTES
 
Accepted for publication March 29, 2005.

1 E-mail: melliott{at}ufl.edu


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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Tuite J. 1969. Plant Pathological Methods: fungi and bacteria. Minneapolis, Minnesota: Burgess Publishing Co. 239 p.

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