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Viability and maturation of Aphanomyces cochlioides oospores

     University of Minnesota, Northwest Research and Outreach Center, Crookston 56716 and Department of Plant Pathology, St. Paul, Minnesota 55108

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Plasmolysis, tetrazolium bromide staining and microscopic appearance were tested for their usefulness in determining viability of oospores of Aphanomyces cochlioides. For comparison, three lethal treatments were employed to contrast the reaction of dead oospores and untreated, presumably viable oospores. Few oospores stained with tetrazolium bromide, even though plasmolysis and microscopic appearance indicated that 85% were viable. Cytoplasm of viable oospores was densely organized and uniformly granular (DOUG), whereas cytoplasm of oospores exposed to lethal treatments was loosely organized and non-uniformly granular (LONG). Dose-response bioassay experiments were conducted with untreated oospores of varying inoculum densities or with mixtures of untreated DOUG and heat-treated LONG oospores in varying proportions. The number of DOUG oospores was correlated (R² = 0.62, P < 0.001) with severity of damping-off of sugar beet seedlings caused by A. cochlioides. Thus, the granular appearance of cytoplasm offered a fast, easy and reliable indicator of viability of A. cochlioides oospores. Tests with newly formed oospores/oogonia showed that >80% harvested at 3–4 d after inoculation of hypocotyls stained with tetrazolium, but by 8–9 d <10% stained, apparently because of declining permeability of the spore wall to tetrazolium as oospores matured.

Key words: oomycete, oospore production, oospore survival, plasmolysis, Saprolegniales, tetrazolium

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The oomycete, Aphanomyces cochlioides Dreschler, is an important cause of damping-off and root rot of sugar beet (Beta vulgaris L.) (Didelot et al 1995, Papavizas and Ayers 1974, Payne et al 1994, Smith 1997, Szymczak and Banaszak 1994, Windels and Lamey 1998). The life cycle of A. cochlioides includes two spore types: short-lived, motile zoospores and long-lived, dormant oospores. In warm, moist soil, root exudates stimulate dormant oospores to germinate (unpubl). Germinating oospores produce hyphae that directly infect the root or produce zoospores that swim through soil water to sugar beet roots, where they encyst, germinate and infect. After infection occurs, the plant pathogen ramifies in the root and eventually kills surrounding tissue, which results in production of disease symptoms. As plant tissues die, A. cochlioides rapidly produces oospores. Overall, A. cochlioides spends only a short time as zoospores and hyphae; most of the time it survives as dormant oospores.

Because oospores are the primary inoculum in the life cycle of A. cochloides, it is critical to have an effective and reliable assay of oospore viability to study the pathogen's survival and to standardize inocula for experimental treatments. Several methods have been developed for producing Aphanomyces oospores (Paternoster and Burns 1996, Parke and Grau 1992) but there is no method to reliably determine their viability, and only a low percentage of oospores germinate in vitro (Papavizas and Ayers 1974).

Visual assays for determining viability in oospore-forming species commonly have included treatment with vital stains such as tetrazolium bromide or with plasmolyzing solutions. Oospores were presumed viable if they stained a rose or lavender color with tetrazolium bromide (Sutherland and Cohen 1983, Jiang and Erwin 1990, Van der Gaag 1994) or if plasmolysis occurred in 4 M NaCl (Mayton et al 2000, Jiang and Erwin 1990, Pittis and Shattock 1994). For either procedure, the assay has been tested by comparing reactions of untreated oospores with oospores subjected to a pretreatment presumed to be lethal, such as immersion in boiling water or by autoclaving. Validating viability of oospores requires correlation between the visual assay and ability of oospores to cause disease. Bioassays, such as the most probable number assay or soil indexing (Pfender et al 1981, Sherwood and Hagedorn 1958), have intrinsic problems. They are labor intensive, require large numbers of oospores and are imprecise. Therefore, they are not suitable for routine estimates of viability of oospore samples. The objective of this research was to identify a simple, accurate visual viability assay applicable to oospores of A. cochlioides.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Oospores were produced in sugar beet hypocotyls of cultivar American Crystal 205 (American Crystal Sugar Company, Moorhead, MN). Seedlings were grown in the greenhouse in 10-cm-diameter plastic pots (25 seeds/pot) containing a pasteurized soil mix (6:6:5:2, v:v:v:v, Waukegan silt loam field soil:sand:peat:composted manure [pH = 7.2], respectively). The greenhouse was naturally lighted at 23 C (14 h) and 16 C at night, but from October through March, was supplemented with 14 h illumination from two, 400-watt high-pressure sodium lamps. Hypocotyls approximately 2-cm in length were excised from 2-wk-old seedlings, surface-treated for 30 s in 0.25% sodium hypochlorite (NaOCl), rinsed twice with sterile distilled water (SDW), and placed in 10-cm diameter petri dishes (two hypocotyls/dish) containing 20 mL SDW. Hypocotyls were inoculated with a 0.4-cm diameter core of A. cochlioides removed from the margin of an actively growing culture on cornmeal agar amended with 50 mg/L each of rifampicin and penicillin G and incubated in the dark at 17–23 C. Oogonia commonly were observed in hypocotyls 1 to 2 d later. In this study, oospore age was based on the length of time between inoculation of hypocotyls and extraction for experiments. To extract oospores, 20 hypocotyls were placed in a 7-mL Tenbroeck tissue macerator filled with sterile distilled water; the plunger was depressed 15 times and the resulting oospore suspension was decanted.

The appearance of untreated oospores was compared to those exposed to lethal treatments. Two replicated experiments were conducted, the first with 8-wk-old oospores and the second with 10-wk-old oospores. In both experiments, three aliquots of oospores were removed from a common oospore suspension and exposed to one of three treatments to cause death: 35% ethanol for 24 h, boiling water for 20 min, or 0.5% NaOCl for 24 h. Control oospores were untreated. Treated and control oospores were maintained at 25 C for 24 h and evaluated for viability by three tests including: (i) plasmolysis in 4M NaCl (J. Jiang and D.C. Erwin 1990), (ii) a tetrazolium bromide stain (0.1% MTT [Sigma, St. Louis, Missouri]), pH 6.5, 35 C for 48 h (Van der Gaag 1994) and (iii) microscopic examination at 400 x magnification. A typical oospore contains granular cytoplasm with refractive bodies (Scott 1961), which we described as densely organized and uniformly granular (DOUG). For each lethal treatment and subsequent viability test, five samples of 50 oospores each were assessed microscopically.

To determine if oospores assessed as dead were infectious, soils were infested with 0, 20, 40 or 60 untreated 5-wk-old oospores/cm³ or with 20, 40 or 60 heat-treated oospores/cm³. The heat treatment consisted of subjecting oospores to 45 C for 24 h, which we previously had found changed all oospores in the suspension to a loosely organized and non-uniform granular (LONG) appearance. Damage occurred while extracting oospores from hypocotyls, and viability of untreated oospores was 59% for the first trial and 76% for the second trial. Because of greater oospore viability in the second trial, total numbers of untreated and heat-treated oospores were reduced to 10, 20 and 40/cm³. For each treatment, five 10-cm diameter pots were filled with unpasteurized greenhouse soil mix infested with oospores of A. cochlioides and planted with 25 sugar beet seeds of cultivar American Crystal 205 at a 1-cm depth. Pots were placed in a greenhouse under natural lighting at 23 C day (14 h) and 16 C at night for 1 wk, until seedlings emerged; they then were moved to a growth chamber set at 28 C, a 16-h photoperiod and 70% relative humidity. Stand counts were made at emergence and every other day thereafter. Dying seedlings were promptly removed and processed in a water assay to identify causal agents (Windels and Nabben-Schindler 1996). The trial was concluded when more than 80% of seedlings had died in any one treatment. The experiment was repeated in a growth chamber following the temperature protocol previously described.

A second dose response test was performed with heat-treated oospores subjected to 40 C for 48 h, rather than 45 C for 24 h as done in the first test. The milder heat treatment resulted in a low percentage of DOUG oospores and therefore provided a more stringent comparison between the visual viability assay and the bioassay of oospore infectivity. For seven soil treatments, unpasteurized greenhouse soil mix was inoculated with heat-killed + untreated control oospores to bring the total concentration of oospores for each treatment to 30/cm³ of soil. By varying the proportion of heat-treated and untreated oospores in the seven soil treatments, final concentrations of living oospores (DOUG, based on microscopic assessment) were 1, 3, 5, 9, 13, 17 and 22/cm³ of soil. For seven additional treatments, soil was inoculated with sufficient untreated control oospores to provide concentrations of 0, 5, 10, 15, 20, 25 and 30/cm³; because 86% of these oospores had a DOUG appearance, the number of putative viable oospores was about 0, 4, 8, 13, 17, 21 and 25/cm³, respectively. For each of the 14 soil treatments, six pots were planted with sugar beet seed as previously described. When the trial was repeated, the percentage of viable oospores varied slightly. Consequently, the range for the mixed oospore series was from 2 to 17 DOUG oospores/cm³ and for the untreated series ranged from 0 to 20 DOUG oospores/cm³.

Regression analysis was applied to the bioassay data. The proportion of dead seedlings out of total emergence for each pot was calculated for each observation day and an area under a disease-progress curve (AUDPC) was calculated. To attain more uniform variances among treatments, the final AUDPCs were divided by the maximum observed AUDPC for the trial and subjected to arc sine square-root transformations. These transformed results, minus the zero control, were examined by regression analysis against the total number of living oospores (as determined by microscopic examination for DOUG) for each treatment series employing the program ARC 1.03, rev Aug, 2000, © 1999–2000, R. D. Cook and S. Weisberg (http://www.wiley.com/mathematics). The difference in slope between the two curves was evaluated by the student's t-test.

To test whether tetrazolium staining reflected oospore age, five hypocotyls of 2-wk-old seedlings of cultivar American Crystal 205 were inoculated as previously described. Segments (3-mm long) were excised from hypocotyls at 3, 5, 7, 9, 11, 14 and 28 d after inoculation and stained with tetrazolium bromide. Segments were examined microscopically at 400 x to determine if oospores had stained a rose to lavender typical of tetrazolium staining. For each segment, 30 oospores were observed within a randomly selected field of view. When the experiment was repeated, segments of hypocotyl were removed and stained 4, 6, 8, 10, 12 and 14 d after inoculation and examined.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In this study, oospore production averaged 8 x 10³/hypocotyl (SD = 6.4 x 10³) and ranged from a low of 2.5 x 10³/hypocotyl in January to a high of 18.3 x 10³/hypocotyl in August. Oospore suspensions produced in this manner contained varied protists but rarely other fungi or oomycetes. The protists had no apparent affect on oospores of Aphanomyces cochlioides, and their feeding and general activity subsided after two to three wk. Oospore suspensions stored at 17–23 C in parafilm-sealed test tubes for more than two years remained viable and lacked fungal contamination although no special precautions were taken to exclude airborne spores while suspensions were prepared.

Results of viability assays for oospores exposed to lethal treatments were not statistically different in both trials, so data were combined. The lethal treatments, however, varied in suitability for determining oospore viability. Oospores exposed to ethanol or boiling water were killed. Oospores exposed to NaOCl resulted in distorted walls that made them unsuitable for evaluation by the plasmolysis and tetrazolium assays but did not impair evaluation by the DOUG assay (Table I).

In the tetrazolium assay, oospores that stained a rose or lavender were designated viable. A low percentage of ethanol-treated and heat-treated oospores stained rose or lavender but these results also occurred in the untreated control where presumably viable oospores were stained by tetrazolium (Table I). The unexpected response of untreated control oospores to tetrazolium suggested that most mature oospores might be impermeable to the stain.

Microscopic examination revealed that cytoplasm of most untreated oospores was densely organized and uniformly granular (DOUG, Fig. 1A) but cytoplasm of all oospores subjected to ethanol or boiling water lacked granules or were loosely organized and non-uniformly granular (LONG, Fig. 1B). The DOUG assay detected a low proportion of viable oospores among those treated with NaOCl. Overall, plasmolysis and DOUG assays gave comparable results for oospore viability (Table I).

View larger version (92K): [in this window] [in a new window]     FIG. 1. Microscopic view of oospores of Aphanomyces cochlioides (scale bars = 10 µm): A.) densely organized and uniform granular (DOUG) appearance of a viable oospore and B.) loosely organized, nonuniform granular (LONG) appearance of a dead oospore

Dose response curves from both trials in the second experiment, with a series of mixtures of LONG and DOUG oospores, were similar, so data from one trial are presented (Fig. 2). Regression analyses showed a significant relationship between number of DOUG oospores/cm³ of soil and transformed amount of disease (Fig. 2). Testing for an interaction between inoculum dose and soil series showed no significant differences in slopes for the heat-treated + untreated oospore soil series versus the untreated oospore series (P = 0.30, data not shown). No seedlings died from Aphanomyces damping-off in the uninoculated control. For the two trials, the resulting AUDPC values ranged from a low of 0.03 for one viable (DOUG) oospore/cm³ of soil to a high 2.69 for 22 viable (DOUG) oospores/cm³ of soil.

View larger version (21K): [in this window] [in a new window]     FIG. 2. Regression line (reconverted to area under the disease-progress curve [AUDPC]) showing the relationship between disease (measured as AUDPC) and concentration of densely organized and uniformly granular (DOUG) oospores of Aphanomyces cochlioides in two soil series (R2 = 0.62, P < 0.001). One series consisted of seven soils infested with untreated oospores with total DOUG oospores concentrations of 0, 4, 8, 13, 17, 21 and 25/cm3. The other series consisted of seven soils infested with varying proportions of heat-treated and untreated oospores to maintain a total concentration of 30 oospores/cm3 with DOUG oospore concentrations of 1, 3, 5, 9, 13, 17 and 22/cm3. Mean values are presented for each treatment

Most newly formed oospores/oogonia stained well with tetrazolium bromide, but older oospores did not. In the first trial, 82% of the oospores/oogonia harvested 3 d after inoculation stained a rose or lavender color (Fig. 3). The percent of oospores/oogonia that stained dropped rapidly thereafter, until 11 d after inoculation, only 3% were stained. Results from the second trial were similar except that the decline in proportion of oospores stained appeared to be delayed by one d.

View larger version (31K): [in this window] [in a new window]     FIG. 3. Percent oospores stained by tetrazolium bromide from 3 to 28 d after inoculation of hypocotyls with Aphanomyces cochlioides in Experiment 1 and from 4 to 14 d in Experiment 2. Each data point is an average based on microscopic examination of 150 oospores

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Tetrazolium bromide did not ascertain viability of oospores as evidenced by a low percentage that stained a pink or lavender whether they were exposed to lethal stresses or untreated. These results explain earlier work where oospores of A. cochlioides and A. euteiches were not stained by tetrazolium (Sutherland and Cohen 1983). Tetrazolium bromide stain likely is a measure of permeability of the oospore wall, and lack of stain might reflect maturation or dormancy. A small but consistent number of oospores stained irrespective of age, which suggested an artifact or the possibility that they remained in an active physiological condition. Perhaps these oospores had broken dormancy and were ready to germinate; this would fit the observation that only a small percent of oospores germinate at any given time (Papavizas and Ayers 1974).

An additional benefit of this research was the development of an easy, rapid method for producing abundant oospores in vivo in excised hypocotyls, a natural substrate for A. cochlioides. Oospore production was excellent with less than four hypocotyls needed, on average, to infest 1000 cm³ of soil at 30 oospores/cm³. Production seemed to be affected by hypocotyl quality. Vigorous seedlings grown in the greenhouse under natural light in August produced seven times more oospores than visibly less vigorous seedlings grown in January. Although we inoculated sugar beet hypocotyls with mycelium, inoculations with as few as 100 zoospores/two hypocotyls also were effective. Oospores produced in hypocotyls coexisted with other microflora, which is typical of natural conditions, and the microflora did not adversely affect oospores. Oospore suspensions maintained for over two years at 17–23 C remained viable and infectious (unpub). Unlike our experience with oospores produced in oatmeal broth (Parke and Grau 1992) that are very susceptible to fungal contaminants, oospore suspensions produced from excised hypocotyls remained free of fungal contaminants without special precautions. Extraction of oospores from hypocotyls with the tissue macerator, however, tended to reduce numbers of viable oospores.

In summary, examination of oospores of A. cochlioides for a densely organized and uniform granular (DOUG) appearance proved to be an effective and accurate measure of viability. This procedure provided results similar to plasmolysis but had advantages over the latter procedure, including clearer visual designations, gentler treatment of oospores and freedom from reagents. The stain, tetrazolium bromide, did not provide a measure of oospore health but instead provided information on impermeability of the oospore to water and water-soluble substances. This impermeability appeared related to oospore maturity and might relate to an oospores' disposition to germinate.

	ACKNOWLEDGMENTS

 
We thank Dr. Kurt Leonard and Dr. James Kurle for their invaluable assistance and advice. This research was financed by the Sugarbeet Research and Education Board of Minnesota and North Dakota, University of Minnesota Northwest Research and Outreach Center and Minnesota Agricultural Experiment Station.

	FOOTNOTES

 
¹ Corresponding author, alanddd{at}hotmail.com

Accepted for publication August 31, 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Dyer, A. T. Articles by Windels, C. E. Search for Related Content PubMed Articles by Dyer, A. T. Articles by Windels, C. E. Agricola Articles by Dyer, A. T. Articles by Windels, C. E.