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Crop Science 43:92-95 (2003)
© 2003 Crop Science Society of America

CROP BREEDING, GENETICS & CYTOLOGY

Partial Resistance to White Mold in a Transgenic Soybean Line

Elroy R. Cober*,a, Sylvie Riouxb, Istvan Rajcanc, Pauline A. Donaldsona and Daina H. Simmondsa

a Agriculture and Agri-Food Canada, ECORC, Building 110, Central Exp. Farm, Ottawa, ON, K1A 0C6 Canada
b Centre de Recherche sur les Grains, Inc., 2700 rue Einstein, bur. D1 300.24A, Sainte-Foy, QC, G1P 3W8 Canada
c Dep. of Plant Agriculture, Crop Sci. Bldg., Univ. of Guelph, Guelph, ON, N1G 2W1 Canada

* Corresponding author (coberer{at}em.agr.ca)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Oxalic acid is an important pathogenic factor for the fungus Sclerotinia sclerotiorum (Lib.) de Bary. An oxalate degrading enzyme, oxalate oxidase (OxO), in transgenic soybean [Glycine max (L.) Merr.] has reduced pathogen growth in indoor seedling studies. The objective of this study was to characterize the response of this OxO transgenic soybean line to white mold under field conditions and also to characterize the agronomic performance of this transgenic line under noninfected conditions. The transgenic line 80(30)-1contains the wheat germin gene gf-2.8 that codes for OxO. This line was compared with 80(30)-9, a sib line which does not contain the gene, as well as resistant and susceptible cultivars. Field tests were conducted at three sites infested with white mold in Ontario and Quebec across 3 yr. The Sainte-Foy site provided the highest infection potential and the transgenic line had a disease severity index (DSI) of 7 compared with 80(30)-9 with a DSI of 46, across 3 yr. Across 2 yr, resistant commercial cultivars had an average DSI of 2 and susceptible cultivars a DSI of 46. Stem inoculations were performed in the field at Elora and the transgenic line was significantly less infected compared with 80(30)-9. In noninfested trials, no significant differences were found between the transgenic, the negative sib, and the parental lines for seed yield, maturity, seed weight, or seed protein and oil content. The transgene provided white mold resistance equivalent to the best commercial cultivars in a white mold susceptible background.

Abbreviations: DSI, disease severity index • OxO, oxalate oxidase • PDA, potato dextrose agar • QTL, qualitative trait loci


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Sclerotinia sclerotiorum is a fungal pathogen which causes sclerotinia stem rot, also known as white mold, in soybean. White mold has become an important soybean disease in parts of Canada, Argentina, and the northern region of the United States (Wrather et al., 1997). Genetic variation for resistance to the disease is limited in commercial germplasm. Breeding for white mold resistance is difficult partly because of low correlation between field and laboratory tests of resistance (Boland and Hall, 1987; Wegulo et al., 1998; Kim et al., 2000) and because resistance is often due to disease avoidance traits rather than physiological resistance (Sutton and Deverall, 1984; Boland and Hall, 1987; Kim et al., 1999; Kim and Diers, 2000). Quantitative trait loci analysis for partial resistance in a ‘S19-90’ x ‘Williams 82’ population revealed several qualitative trait loci (QTL) associated with escape mechanisms; however, only one QTL was associated with physiological resistance (Kim and Diers, 2000). Arahana et al. (2001) examined five populations for resistance to white mold using a detached leaf assay and found a number of QTL. Unfortunately, QTL reported in these two studies had small effects, each explaining 10% or less of the variation, making breeding with these loci difficult.

Most infections of soybean by S. sclerotiorum originate from colonization of flower petals by airborne ascospores. Fungal invasion of nodes and stems often results in plant death (Grau, 1988). Invasion of healthy plant tissue requires fungal secretion of oxalic acid, the probable cause of the lesions formed in advance of the invading fungal hyphae (Lumsden and Dow, 1973). Hypovirulent fungal isolates produced less mycelium and oxalic acid compared with virulent isolates (Zhou and Boland, 1999). Oxalic acid has been shown to be an important pathogenicity factor by using S. sclerotiorum mutants deficient in production of oxalic acid (Godoy et al., 1990). Field resistance in some genotypes has been correlated with laboratory resistance to oxalic acid (Wegulo et al., 1998). Oxalic acid was recently shown to suppress the oxidative burst in host plants, thus disabling a major plant resistance system (Cessna et al., 2000). Oxalic acid also lowers extracellular pH, thereby optimizing fungal lytic enzyme function (Maxwell and Lumsden, 1970; Marciano et al., 1983) and weakens cell walls via chelation of calcium from wall calcium pectate (Bateman and Beer, 1965) and inhibition of {theta}-diphenol oxidase (Marciano et al., 1983; Ferrar and Walker, 1993).

An obvious defense strategy against S. sclerotiorum is the use of a transgenic soybean which produces an enzyme, in this case wheat germin, an OxO (oxalate:oxygen oxidoreductase, EC 1.2.3.4), that degrades oxalic acid. Oxalate oxidase catalyzes the oxidation of oxalic acid by molecular oxygen to carbon dioxide and hydrogen peroxide. Transgenic soybean homozygous for the OxO gene showed greatly reduced disease progression and lesion length following cotyledon and stem inoculation with S. sclerotiorum in growth cabinet tests (Donaldson et al., 2001).

The use of OxO to degrade oxalic acid may serve multiple roles: destroying the fungal toxin and at the same time generating H2O2 (Lane, 1994, 2000); and maintaining the oxidative burst (Cessna et al., 2000). The production of H2O2 is important because it plays a key role in plant defense as an inducer of cellular protection genes and the hypersensitive response (Levine et al., 1994; Tenhaken et al., 1995), and it is a substrate for peroxidase-mediated cross-linking of hydroxyproline rich cell wall glycoproteins that strengthen cell walls (Bradley et al., 1992).

The objective of this study was to characterize the response of an OxO transgenic soybean line to white mold under field conditions and to characterize the agronomic performance of this line under noninfected conditions.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The transgenic line 80(30)-1 has been described by Donaldson et al. (2001). Briefly, 80(30)-1 resulted from Agrobacterium-mediated transformation and contains the wheat germin gene gf-2.8 that codes for OxO, which degrades oxalic acid, in the genetic background of ‘AC Colibri’. An OxO negative line, 80(30)-9, and 80(30)-1 were both selected from the same T0 plant which segregated in a Mendelian fashion. 80(30)-9 was used as a control line. A number of cultivars were selected as resistant and susceptible controls based on white mold infection in white mold tests conducted at Ottawa in 1996 and 1997. In these tests, the mean percentage infected stems were: ‘Nattosan’, 87%; AC Colibri, 40%; ‘Maple Arrow’, 13%; and ‘OAC Salem’, 10%; with an LSD0.05 of 7%.

Field tests were conducted at Ottawa and Elora, ON, and Sainte-Foy, QC, Canada during 1998, 1999, and 2000. The soil at the Ottawa site is a Granby sandy loam (Orthic Humic Gleysol in Canadian soil classification; Humaquept in American Classification system), the soil at the Elora site is a Woolwich silt loam (Brunisolic Gray Brown Luvisol in Canadian soil classification; Hapludalf in American Classification system), and the soil at the Sainte-Foy site is a St. Nicolas gravelly loam (Orthic Humo-Ferric Podzol in Canadian soil classification; Haplorthod in American Classification system). At Ottawa, the tests were grown in a naturally infested white mold nursery which was irrigated by overhead sprinkling for 45 min daily from first flower until the beginning of seed filling. At Elora, the tests were grown in a naturally infested field, which was irrigated by overhead sprinkling during the middle of the afternoon for 1 hr daily from first flowering until the beginning of seed filling. At Sainte-Foy, the tests were grown in a nursery artificially infested with sclerotia of S. sclerotiorum. The nursery was irrigated with overhead sprinkling as needed to keep the soil surface wet, from the first inoculation ({cong}2 wk before the beginning of flowering) until symptom observations were complete (beginning of pod maturity). In 1998, the tests were single row plots, 2- to 4-m long, with 24 replications in a completely randomized design. In 1999 and 2000, the tests were four row plots, 2- to 5-m long, with four replications in a randomized complete block design. Row spacing was 400 mm (200 mm in 2000) at Ottawa, 500 mm at Elora, and 180 mm at Sainte-Foy. The plots were planted at a rate of 55 seeds m-2 at Ottawa, 58 seeds m-2 at Elora, and 89 seeds m-2 at Sainte-Foy. The trials were planted on 2 June 1998, 4 June 1999, and 31 May 2000 at Ottawa, on 30 May 1998 and 7 June 1999 at Elora, and 29 May 1998, 31 May 1999, and 29 May 2000 at Sainte-Foy. The parental and some check cultivars were also grown in the white mold nursery at Ottawa in 1996 and 1997.

Plants were rated for DSI (Grau et al., 1982; Kim and Diers, 2000) at the beginning of leaf senescence (a few days before the appearance of a pod with mature color). Thirty plants in a plot were rated on a scale of 0 to 3, where 0 = no symptoms, 1 = lesions on lateral branches only, 2 = lesions on main stem but little effect on pod fill, and 3 = lesions on main stem resulting in plant death and poor pod fill. Disease severity index was calculated for each plot using the formula

This results in a DSI of 0 for no plants rated infected and a DSI of 100 for all rated plants killed by white mold. The percentage of rated plants with any infection was also calculated.

At Elora, plant stems were also artificially inoculated using the following procedure. Sclerotia were collected from the field in the year prior to inoculation. Sclerotia were sterilized with 2.5% sodium hypochlorite solution. The sclerotia were then placed in the middle of a 100- by 15-mm Petri dish containing {cong}30 mL of 10% potato dextrose agar (PDA). The Petri dishes were maintained at room temperature (18 to 22°C) under dim light for 5 d while thick mycelium growth developed. Bulk culture was made by transferring a piece from the growing edge of the mycelium and agar to the center of a new Petri dish containing 10% PDA. After 3 d, four pieces of agar and mycelium were transferred into a 500-mL Erlenmeyer flask. The flask contained 150 g of imbibed barley (Hordeum vulgare L.) seed and 150 mL of distilled water and was autoclaved at 121°C for 20 min prior to the inoculation. The cultures in Erlenmeyer flasks were placed at 25°C in dim light for 14 to 21 d.

At flowering time, a hole was made in the plant stem 150 to 200 mm from the soil surface (second to third internode) using a scratch awl. An infected barley seed from culture was placed in the hole and left in place. Ten plants per plot were inoculated. After 1 wk, the lesion caused by the pathogen was measured as it extended up or down from the wound (extension was usually symmetrical). The scoring scale that was used in 1998 was 1 to 3 (1 = lesion from 0- to 20-mm long; 2 = lesion >20- to 50-mm long; 3 = lesion of >50 mm usually resulting in plant death). In 1999, the scale was expanded from 0 to 4 (0 = only the wound is visible with a thin black circle around it but no tanning of the surrounding tissue; 1 = 0- to 20-mm-long tanned lesion around wound; 2 = >20- to 50-mm-long tanned lesion; 3 = >50-mm lesion coupled with whitening of the tissue but plant still alive; and 4 = >50 mm lesion resulting in complete death of plant). In both years, the average of 10 plants was calculated and expressed as an inoculation severity index for each plot.

In 1999 and 2000, trials were also grown in noninfested fields without irrigation at Ottawa and Sainte-Foy. At Ottawa, the trials were grown in 5-m, four-row plots with 400-mm row spacing. Plots were seeded at a rate of 50 seeds m-2 on 28 May 1999 and 21 May 2000. At Sainte-Foy, the trials were grown in 5-m, four-row plots with 180-mm row spacing. Plots were seeded at a rate of 67 seeds m-2 on 31 May 1999 and 29 May 2000. Trials were arranged in randomized complete block designs. Four rows were combine harvested and yields were adjusted to 130 g kg-1 moisture. The following observations were taken from the center two rows: date of maturity, when 95% of pods reached mature color; and main stem height at maturity. Seed was evaluated for 100-seed weight, and seed protein and oil content determined on a whole seed dry matter basis by near infrared transmittance spectroscopy. Analyses of variance were performed using the GLM procedure of SAS (SAS Institute, Cary, NC).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
In infected fields, the transgenic line was significantly less infected with white mold than the negative sib line (Tables 1 and 2). The Sainte-Foy site provide the highest infection with the susceptible cultivar Nattosan having a DSI of 61 across 2 yr (Table 2). At Sainte-Foy, across 3 yr, the transgenic line 80(30)-1 had an average DSI of 7 while the negative sib 80(30)-9 had an average DSI of 46 (Tables 1 and 2). The resistant cultivars Maple Arrow and OAC Salem had a DSI of 2 during 1999 and 2000. Disease pressure was low at Ottawa in 1998 (Table 1) and moderate in 1999 where the transgenic line and negative sib had a DSI of 3 and 14 (LSD0.05 = 7) respectively. The infected trials at Ottawa and Elora were lost due to flooding in 2000. At Sainte-Foy, the transgene reduced the DSI {cong}29 units compared with the largest QTL reported by Kim and Diers (2000) which reduced DSI about six units. While the transgenic line 80(30)-1 was not immune to white mold infestation, the transgene, in a background which is quite susceptible to white mold, provided a resistance level equivalent to the current best short-season cultivars.


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  		Table 1. Disease severity index, and percentage plants infected for the transgenic soybean 80(30)-1 and control 80(30)-9 from trials in white mold infested nurseries at Sainte-Foy, Elora, and Ottawa, Canada, in 1998.

 

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  		Table 2. Mean disease severity index and seed yield for transgenic soybean 80(30)-1 and control cultivars from trials in a white mold infested nursery at Sainte-Foy, Canada, in 1999 and 2000.

 
Individual plants were stem inoculated in the field at Elora. Using an index to score disease severity, the transgenic line 80(30)-1 was significantly less affected compared with the negative sib line 80(30)-9 (Table 3). In 1999 all other cultivars, both resistant (Maple Arrow and OAC Salem) and susceptible (Nattosan and ‘OAC Shire’), were severely infected. This direct infection of stem tissue appears to negate possible avoidance measures in the resistant cultivars. The data indicate that the transgene provides physiological resistance to the pathogen.


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  		Table 3. Inoculation severity index from soybean plants inoculated in the field at Elora, Canada, in 1998 and 1999.

 
Donaldson et al. (2001) proposed that at least three related mechanisms may contribute to increased resistance in this transgenic line: (i) disarming of the primary action of the oxalate toxin on cell walls, (ii) expression of latent resistance through removal of oxalate which suppresses the oxidative burst (Cessna et al., 2000), and (iii) triggering of pathogen defense responses by H2O2 (Levine et al., 1994). In a transgenic line, the combination of physiological resistance (i and iii previous) provided by the transgene could be combined with field avoidance traits and possibly latent resistance (ii previous). The discovery of latent resistance will involve the incorporation of the transgene into a large number of genetic backgrounds with comparisons made between positive and negative near-isogenic lines. Additionally, a patent by Pioneer Hi-Bred (Bidney et al., 2000) reports "The synergistic effect of expression of a hydrogen peroxide/reactive oxygen species producing enzyme or an oxalate degrading enzyme in a resistant background gives significant and unexpectedly high resistance to the pathogens."

We also evaluated the transgenic and control lines in noninfested sites to test for possible effects the transgene might have on agronomic performance. Across four site-years, no significant differences were observed between the transgenic line, and the negative sib line for seed yield, maturity, seed weight, and seed protein and oil content (Table 4). In 2000, the parental cultivar AC Colibri was also included as a control and no agronomic differences were found between AC Colibri, the transgenic line, and the negative sib line (Table 4). There does not seem to be any yield reduction in this transgenic line.


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  		Table 4. Mean yield and agronomic characteristics of the transgenic soybean 80(30)-1, the null sib line 80(30)-9, and check cultivars grown in noninfested fields at Ottawa and Sainte-Foy, Canada.

 
In summary, this OxO transgenic event provided white mold resistance levels equivalent to the best available resistant cultivar in a background that is as susceptible to white mold as the worst cultivars. We could not detect any agronomic penalty associated with this transgenic event.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
ECORC Contribution no. 02-09.

Received for publication January 28, 2002.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 




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