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© 2009 Plant Management Network.
Accepted for publication 1 December 2008. Published 28 January 2009.


Development of a Rapid Method Using Oxalic Acid to Assess Resistance Among Hosta Cultivars to Petiole Rot Caused by Sclerotium rolfsii var. delphinii


Zhihan Xu, Hampton Road AREC, Virginia Tech, Virginia Beach, VA 23455 (previously of Iowa State University); Mark L. Gleason and Daren S. Mueller, Iowa State University, Ames IA 50011


Corresponding author: Mark L. Gleason. mgleason@iastate.edu


Xu, Z., Gleason, M. L., and Mueller, D. S. 2009. Development of a rapid method using oxalic acid to assess resistance among hosta cultivars to petiole rot caused by Sclerotium rolfsii var. delphinii. Online. Plant Health Progress doi:10.1094/PHP-2009-0128-01-RS.


Abstract

A rapid assay was developed to assess hosta cultivars for resistance to petiole rot caused by Sclerotium rolfsii var. delphinii. Leaves of greenhouse-grown hosta (Hosta kikutii and Hosta spp. cultivars Munchkin, Lemon Lime, Tardiflora, Pearl Lake, Zounds, Gold Drop, Halcyon, and Honeybells) were treated with 20 ml of oxalic acid (50 mM) on a cotton swab, then incubated at 100% relative humidity and 27°C. After 4 days, incidence of leaves with lesions was evaluated. Cultivars Munchkin, Lemon Lime, and Tardiflora had a relatively high incidence of leaves with lesions, whereas Gold Drop and Halcyon had much lower incidence. These results were generally consistent with those of field and greenhouse cultivar screening tests in which whole plants were inoculated with the pathogen and rated for disease incidence. Additional screening methods, including spray application of either oxalic acid or mycelial fragments of S. rolfsii var. delphinii, were not as repeatable or simple to conduct as the cotton swab assay. The cotton swab method showed potential to accelerate identification of highly resistant hosta cultivars, and thereby aid efforts to breed resistance to petiole rot.


Introduction

Petiole rot, incited by Sclerotium rolfsii and S. rolfsii var. delphinii, causes substantial losses in hosta (Hosta kikutii and Hosta spp.) plantings in the United States (Fig. 1) (5). The common name was changed from crown rot (5) to petiole rot because another hosta disease, caused by Fusarium spp., was also designated crown rot (16). Previously known only from the southern United States (11,13,15), hosta petiole rot was reported in the midwestern United States with increasing frequency during the 1990s (5). Petiole rot is difficult and expensive to control in nurseries and landscapes because the causal fungi can survive in the soil for many years and can spread from hosta to hundreds of annual and perennial plant species. Available information suggests that S. rolfsii var. delphinii is the predominant petiole rot pathogen in the northern United States, whereas S. rolfsii is predominant in southern states (2,18).


 

Fig. 1. Symptoms and signs of hosta petiole rot.

 

Using resistant cultivars can be an effective method of managing diseases incited by these fungi (11). Identifying hosta cultivars and species with high levels of resistance to petiole rot would benefit breeders, growers, marketers, and hosta enthusiasts by providing a durable, cost-effective management option. Information on levels of genetic resistance to S. rolfsii and S. rolfsii var. delphinii among the more than 4,000 named hosta cultivars is scant. In a greenhouse evaluation of 18 hosta cultivars, Edmunds et al. (6) inoculated plants by placing a carrot disk infested with mycelium of S. rolfsii var. delphinii at the base of each plant. The cultivars Halcyon and Zounds displayed a high level of resistance, whereas Lemon Lime, Munchkin, Nakaiana, Platinum Tiara, and Tardiflora developed severe symptoms. However, this assay was impractical for screening large numbers of hosta cultivars because it was time-consuming (about 3.5 months), expensive, and labor-intensive.

Techniques used to identify resistance to Sclerotinia sclerotiorum, the fungus that causes white mold in numerous crops, may have potential for adaptation to screening for petiole rot resistance. Like S. rolfsii and S. rolfsii var. delphinii, Sclerotinia sclerotiorum releases oxalic acid, which overcomes host defenses during the infection process. Screening techniques for S. sclerotiorum include detached leaf inoculation, an oxalic acid resistance test, and stem inoculation of intact seedlings in controlled environments (8,9,10,12,17,19). Compared to resistance evaluations on intact plants in the field or greenhouse, a detached tissue assay has several potential advantages. For example, a detached tissue assay could yield results in a relatively short time – a few days compared to months for intact plants – and requires much less space than those using intact plants. An additional benefit is that rare breeding stock and expensive plants could be conserved rather than destroyed by the assay.

Oxalic acid can combine with calcium in plant tissues, removing it from association with pectic compounds in plant cell walls, lowering cell wall pH, and thereby facilitating activity of endopolygalacturonase and cellulase (4,7). Mechanisms of resistance to S. rolfsii or S. rolfsii var. delphinii are unknown, but breaking down or excluding oxalic acid could reduce the pathogenicity of S. rolfsii (13). Wegulo et al. (17) developed an oxalic acid assay to screen soybean resistance to stem rot caused by S. sclerotiorum in which the excised ends of soybean petioles were placed in test tubes containing 40 mM oxalic acid. Kolkman and Kelly (9) found that oxalic acid was useful for assessing resistance of common bean to S. sclerotiorum. An assay using oxalic acid rather than fungal inoculum possesses practical advantages because it could be conducted more rapidly and cheaply, and require less equipment and expertise.

The objective of the present study was to develop an oxalic acid-based assay to screen hosta cultivars for resistance to petiole rot that is simpler and faster than whole-plant inoculation methods.


Assess Hosta Cultivars for Resistance to Petiole Rot

Isolate. Cultures of S. rolfsii var. delphinii isolate Srd1, obtained from a symptomatic hosta in Ames, Iowa, were grown on potato dextrose agar (PDA) (Difco Laboratories, Detroit, MI) in 9-cm-diameter sterile petri dishes under intermittent light at room temperature (22 to 25°C).

Cultivar selection. Hosta cultivars or species used in the present trials (Nakaiana, Munchkin, Lemon Lime, Tardiflora, Pearl Lake, Zounds, Gold Drop, Halcyon, Snow Mound, Honeybells, and H. kikutii) exhibited a range of resistance to petiole rot in previous greenhouse inoculation trials (6). Halcyon was highly resistant, whereas the other cultivars ranged from very susceptible to moderately resistant. Single-eye crowns were obtained from Wade and Gatton Nursery (Bellville, OH).

Plant maintenance. Single-eye plants for each cultivar were planted in 15-cm-diameter plastic pots in a potting mix (50% peat : 30% perlite : 20% steamed soil) in a greenhouse at Iowa State University. Plants were grown at 23 to 25°C under high-intensity sodium vapor lamps with a 14-h light/10-h dark regime. Plants were watered weekly and fertilized (N20-P4.4-K16.6, 100 ppm nitrogen) monthly. Flower stalks were removed as soon as they appeared.

Assessment of resistance in the field. In order to validate results of the Edmunds et al. (6) greenhouse screening trial, seven hosta cultivars (Nakaiana, Munchkin, Lemon Lime, Zounds, Halcyon, Snow Mound, and Honeybells) used in that study were tested outdoors for resistance to petiole rot from 13 May to 7 August 2005 and 23 May to 20 August 2006.

Single-eye hosta plants were established in a naturally shaded field plot at the Iowa State University Horticulture Farm. Composted hardwood bark mulch was used to maintain soil moisture. In each year, the study was set up as a randomized complete block design with six replications. Within each replication, there were three plants per cultivar; petiole bases of two plants of each cultivar were inoculated with carrot disks infested with S. rolfsii var. delphinii on 4 July and 11 July in 2005 and 2006, respectively (18). Moistened sterile cotton balls covered the carrot disks to prevent inoculum from drying out. Infested carrot disks and cotton balls were pinned to the soil in order to prevent inoculum from losing contact with the host plants. Control plants had non-infested carrot disks covered by cotton balls that were treated as described above.

Disease severity was assessed as percent symptomatic petioles. Beginning 4 days after inoculation, plants were rated every 5 days until 7 August 2005 and 20 August 2006. Area under the disease progress curve (AUDPC) was calculated (3). The Tukey-Kramer test was used to compare means of AUDPC. Spearman’s coefficients of rank correlation were used to measure the correlation between ranks of cultivars response in greenhouse trial by Edmunds et al. (6) and ranks of cultivars in field trials.

In field trials, cultivars Nakaiana, Munchkin, and Lemon Lime were most susceptible to petiole rot, Zounds showed moderate resistance, and Halcyon, Snow Mound, and Honeybells were the most resistant (Table 1). Spearman’s coefficients of rank correlation between the greenhouse trial by Edmunds et al. (6) and field trials were 0.57 (nonsignificant, P = 0.18) for the 2005 trial and 0.83 (significant, P < 0.05) for the 2006 trial, indicating that results from the field trials were generally consistent with those of greenhouse inoculation trials (6).


Table 1. Area under the disease progress curve (AUDPC) of hosta cultivars for field trials in 2005 and 2006, and a previous greenhouse trial (6), incidence of leaves with lesions of hosta cultivars in spray-applied oxalic acid trial, and mean incidence of leaves with lesions of hosta cultivars in cotton swab oxalic acid trials.

Cultivar Field trials Greenhouse trials (6) Oxalic acid assays
Spray-applied Cotton swab
AUDPC (2005)t AUDPC (2006)t AUDPC Incidence of leaves with lesions (%)u Incidence of leaves with lesions (%)v
Nakaiana   2607 aw   2012 aw   1215 ax
Munchkin 2490 a 2137 a 1228 a    91.7 abw  75.0 aw
Lemon Lime 2460 a 1833 b 1402 a 100.0 a    71.9 a
Tardiflora y   1142 ab    66.7 abc 62.5 a
H. kikutiiz    66.7 abc 40.6 b
Pearl Lake   810 bc    58.3 bcd 34.4 b
Zounds 1520 b 412 c 33.3 d   31.3 bc
Gold Drop 611 c 25.0 d   12.5 cd
Halcyon 1020 c 638 c   54 d 25.0 d   0.0 d
Snow Mound  972 c 751 c 447 c
Honeybells  795 c   506 cd 416 c   50.0 cd  37.5 b

 t AUDPC of hosta cultivars for field trials. Plants were rated every 5 days, starting 4 days after inoculation, until 7 August 2005 and 20 August 2006.

 u Percent leaves with lesions 4 days after treatment.

 v Percent excised leaves with lesions 4 days (Trial 1) and 5 days (Trial 2) after treatment with oxalic acid in droplets on cotton swab on 9 December 2007 and 16 January 2008; Means shown combined data for both trials.

 w Means followed by the same letters are not significantly different at P = 0.05 according to the Tukey-Kramer test.

 x Means followed by the same letters are not significantly different at P = 0.05 according to the least square difference (LSD) test (6).

 y Not available.

 z Species Hosta kikutii.


Spray-applied oxalic acid assay. In separate trials on 24 October and 21 November 2007, 6- and 10-week-old hosta leaves, respectively, of H. kikutii and Hosta. spp. cultivars Munchkin, Lemon Lime, Tardiflora, Pearl Lake, Zounds, Gold Drop, Halcyon, and Honeybells were arbitrarily chosen, excised from intact greenhouse-grown plants, using scissors that were surface sterilized between each plant sampled, and placed on hardware cloth. The excised base of each petiole was covered with cling-type plastic wrap in order to prevent oxalic acid from coming into contact with it. Fifty ml of oxalic acid (50 mM) (Fisher Scientific, Fair Lawn, NJ) per replication were sprayed evenly on excised leaves using a hand-trigger sprayer, and non-treated control leaves were sprayed with SDW. Sprayed leaves were immediately placed in a dew chamber (100% relative humidity) at 27°C. The trials were set up as a randomized complete block design with three replications. Each replication consisted of four petioles for each of three replications of each cultivar. The incidence (%) of leaves with lesions, calculated by counting the number of leaves with lesions and dividing by the total number tested (×100), was determined 4 days after treatment. The Tukey-Kramer test was used to compare means of incidence of leaves with lesions among cultivars. Spearman’s coefficient of rank correlation was used to measure the correlation between ranks of cultivars and species response in greenhouse trial by Edmunds et al. (6) and the spray-applied oxalic acid trial.

Lesions appeared on most cultivars within 2 days after treatment of 6-week-old petioles and leaves, but cultivar resistance could not be differentiated based on the incidence of leaves with lesions (P = 0.15). When 10-week-old tissues were used, however, there were significant differences (P < 0.05) among cultivars and species in incidence of leaves with lesions 4 days after treatment (Table 1). Cultivars Pearl Lake, Zounds, Gold Drop, Halcyon, and Honeybells had the lowest incidence of leaves with lesions, whereas Lemon Lime and Munchkin had the highest incidence.

Spearman’s coefficient of rank correlation between the greenhouse trial by Edmunds et al. (6) and the spray-applied oxalic acid trial was 0.77 (significant, P < 0.05).

Cotton swab oxalic acid assay. In separate trials on 5 December 2007 and 11 January 2008, 12- and 17-week-old hosta leaves, respectively, of H. kikutii and cultivars Munchkin, Lemon Lime, Tardiflora, Pearl Lake, Zounds, Gold Drop, Halcyon, and Honeybells were arbitrarily chosen, excised with sterile scissors at the base of the crown, and placed on hardware cloth. The tip of a cotton swab was placed at the leaf-petiole junction. Twenty ml of oxalic acid (50 mM) was dispensed onto the tip of the swab with a pipette (Fig. 2). Non-treated control petioles received 20 ml of SDW on swab tips. The trials were set up as a randomized complete block design with four replications. Immediately after treatment, excised tissues and hardware cloth were placed in a dew chamber at 100% relative humidity and 27°C. Incidence (%) of leaves with lesions was determined 4 (Trial 1) and 5 days (Trial 2) after oxalic acid treatment. Analysis of variance was conducted using the SAS PROC Mixed procedure (SAS Institute Inc., Cary, NC). The Tukey-Kramer test was used to compare means of incidence of leaves with lesions among cultivars. Spearman’s coefficient of rank correlation was used to measure the correlation between ranking of cultivar and species response in the earlier greenhouse trial (6) and the cotton swab oxalic acid trial.


 

Fig. 2. Excised leaf (cv. Tardiflora) showing cotton swab onto which a droplet of oxalic acid was dispensed.

 

In both trials, lesions appeared on leaves within 5 days, beginning where the tip of the cotton swab was located. Although results from two runs of the experiment were significantly different (P < 0.05), the F value for interaction between cultivars and experiments was not significant, so results from both experiments were combined. In both trials, cultivars Munchkin, Lemon Lime, and Tardiflora were highly susceptible to oxalic acid, whereas Halcyon and Gold Drop were highly resistant (Table 1; Fig. 3).


 

Fig. 3. Excised leaves of a susceptible cultivar (Munchkin, left) and a resistant cultivar (Gold Drop) after treatment with oxalic acid on cotton swabs, followed by 3 days of incubation at 100% relative humidity and 27°C.

 

Spearman’s coefficient of rank correlation between the cultivar ratings in the greenhouse trial (6) and cotton swab oxalic acid trial was significant (0.79; P < 0.05).


Conclusions and Recommendations

Results from the oxalic acid trials suggest that this approach can dramatically speed up identifying hosta cultivars that are highly resistant to petiole rot. These results are also encouraging evidence that screening methods under controlled conditions can accurately represent cultivar resistance to petiole rot in the "real world," but much more efficiently. Cultivar resistance determined by either spray or cotton swab application of oxalic acid on excised leaves was generally similar to that determined by whole-plant inoculation trials of Edmunds et al. (6) based on Spearman’s coefficients, but required only a few days compared to at least 3.5 months for greenhouse trials using the whole-plant inoculation method. The oxalic acid assays are simple enough to be carried out without specialized laboratory equipment or expertise.

Results of field trials generally validated the cultivar resistance ratings obtained in greenhouse trials by Edmunds et al. (6), in which Halcyon was the most resistant cultivar and Zounds, Snow Mound, and Honeybells were moderately resistant. The result indicated that the greenhouse screening trials method of Edmunds et al. (6) was a valid predictor of cultivar resistance in the field. Greenhouse inoculation assays have an advantage over field trials in that greenhouse temperature and humidity can be controlled to insure conditions favorable for petiole rot development, whereas disease occurrence in the field is more erratic.

Preliminary results in the spray-applied oxalic acid trial suggested that plant age could affect the speed with which leaves developed symptoms. Based on observations in this study, discrimination of cultivar resistance may be more effective with 10-week-old than with 6-week-old petioles or leaves.

Several precautions are advisable when using the oxalic acid assays. A dual-cartridge respirator with face shield should be used for spray application of oxalic acid, since inhaling the vapor can be harmful. No respirator is needed for the cotton-swab assay, but latex gloves should be worn because oxalic acid is a severe skin irritant.

Current management techniques for petiole rot include fungicide application and cultural techniques such as sanitation (11). However, labeled fungicides are not consistently effective and some are human carcinogens (11). Cultural management techniques, such as soil removal and replacement, are labor-intensive and usually impractical. Identifying more cultivars with high levels of resistance will benefit the entire hosta industry by providing a durable, cost-effective option to combat this highly destructive and difficult-to-manage disease. Because S. rolfsii and S. rolfsii var. delphinii also attack hundreds of species of perennial plants, an efficient screening technique for hosta will likely be applicable for screening against these fungi on other herbaceous perennials.

Further research should include applying oxalic acid methods on excised leaves of field-grown rather than greenhouse-grown plants. This modification would benefit breeders who wished to screen their own collections of field-grown plants. Future studies should also determine whether the oxalic acid test on excised leaves could be made even simpler and cheaper by replacing a laboratory dew chamber with much cheaper sealed plastic crispers containing moist paper towels, providing an even more practical and low-cost option for screening hosta cultivars.


Acknowledgments

This study was funded by grants from the Iowa Nursery and Landscape Association Research Committee, The Perennial Plant Association, and The Fred C. Gloeckner Foundation. We thank Anna Peterson for help with statistical analysis, Anne Dombroski, Alex Carlson, Nick Zdorkowski, Edwin Han, Carolina Arce, Adam Sisson, Nenad Tatalović, Alicia Owen, and Rosalee Coelho for technical assistance, and Dave Volkers for maintaining plants in the greenhouse.


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