Saccharin-induced systemic acquired resistance against ...

Crop Protection 30 (2011) 726e732

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Crop Protection

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Saccharin-induced systemic acquired resistance against rust (Phakopsora
pachyrhizi) infection in soybean: Effects on growth and development

Pratibha Srivastava a,*, Sheeja George a, James J. Marois a, David L. Wright a, David R. Walker b

a University of Florida, North Florida Research and Education Center, Quincy, FL 32351, USA
b USDA-ARS, Soybean/Maize Germplasm, Pathology and Genetics Research Unit, Urbana, IL 61801, USA

article info abstract

Article history: We examined the effect of saccharin on the systemic acquired resistance (SAR) response of soybean to
Received 24 October 2010 the fungus Phakopsora pachyrhizi, the causal agent of soybean rust. Plants were grown hydroponically in
Received in revised form half-strength Hoagland’s solution and were challenged with the pathogen 1, 5, 10 and 15 d after treat-
7 February 2011 ment with 3 mM saccharin applied either as a foliar spray or a root drench at the 2nd trifoliate (V3) and
Accepted 14 February 2011 early reproductive (R1) stages. Plants were destructively harvested and assessed for visible rust symp-
toms 2 wk after inoculation. Mode of saccharin application was a significant factor influencing the
Keywords: severity of rust infection. Saccharin applied as a root drench was more effective than the foliar spray
Defense mechanisms treatment at inducing SAR, with increased resistance observed 1 d after application. Systemic protection
Soybean rust against rust infection was still apparent 15 d after application of saccharin as a root drench. In contrast,
Phakopsora pachyrhizi foliar treatment with saccharin did not increase systemic protection until 15 d after treatment. When
Systemic acquired resistance systemic protection was induced by the application of saccharin in either manner, there was no signif-
Saccharin icant reduction of plant growth, except when plants were inoculated 15 d after the saccharin application
as a root drench at the R1 stage of development.

Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction attack or slow down pathogen growth by mobilizing a variety of
biochemical and molecular defenses (Bowles, 1990).
Systemic acquired resistance (SAR) is a broad-spectrum defense
system that is activated in plants upon challenge by certain patho- Establishment of SAR requires an endogenous increase in SA
gens and in response to other environmental stimulants. SAR is levels (Ryals et al., 1996; Sticher et al., 1997; Dempsey et al., 1999)
effective predominantly against biotrophic pathogens, and is and its onset is associated with the expression of SAR genes (Ryals
controlled by a signaling pathway that depends on accumulation of et al., 1996), some of which encode PR proteins (Ryals et al., 1996;
salicylic acid (SA) (Walters et al., 2009). Many instances have been Sticher et al., 1997; Dempsey et al., 1999). Some PR proteins
reported in which pathogen infection of the lower leaves of a plant display antimicrobial activity in vivo (Van Loon and Van Strien,
induced a resistance response in the upper leaves (Kuc, 1982). 1999) but their actual role in SAR remains uncertain. Activation of
Following induction of SAR, the plant is generally resistant to a wide SAR by pathogens has been effective in several plantepathogen
range of pathogens for a period of weeks or months (Ward et al., interactions, i.e., common bean infected by Colletotrichum linde-
1991). The induction of SA signaling and SAR is associated with muthianum (Dann and Deverall, 1995), Arabidopsis infected by
accumulation of pathogenesis related (PR) proteins such as beta-1,3- avirulent pathotypes of Pseudomonas syringae (Alvarez et al., 1998)
glucanases, thaumatin, chitinases and PR1, which are thought to and pea infected with an avirulent strain of Pseudomonas syringae.
contribute to resistance (Van Loon, 1997). Many of the PR proteins
have antimicrobial activity in vitro, but their roles in the establish- Plant defense responses are a result of a complex network of
ment of SAR are unclear. However, they serve as molecular indicators signaling events that involves the interplay of kinases, hormones,
for the onset of the defense response (Van Loon, 1997; Durrant and and reactive oxygen species (ROS), leading to reprogramming of the
Dong, 2004). Following the activation of SAR, plants resist pathogen plant transcriptome and the production of defensive compounds to
affect resistance. Modifications of signaling components can
* Corresponding author. Tel.: þ1 850 875 7152; fax: þ1 850 875 7188. expedite defense activation upon pathogen attack, thus improving
E-mail address: pratibha@ufl.edu (P. Srivastava). the chances of the plant to successfully respond to current and
future encounters with pathogens. In addition to protection
0261-2194/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. conditioned by genes, plant resistance can also be improved by the
doi:10.1016/j.cropro.2011.02.023 use of nontoxic chemical substances that elicit the activation of

P. Srivastava et al. / Crop Protection 30 (2011) 726e732 727

natural defense mechanisms (Conrath et al., 2002; Kohler et al., VII cultivar developed at the University of Georgia (Boerma et al.,
2002). Knowledge about plantepathogen interactions could lead 1997). Both of these cultivars are susceptible to soybean rust,
to solutions for achieving broad-spectrum protection, long-lasting including isolates from Quincy, FL (unpublished data). Seeds from
effects and reduction in pesticide use. the two cultivars were germinated in a medium containing peat
and perlite and maintained in a greenhouse at 75 Æ 2 C and 80%
A number of compounds have been identified to induce SAR, humidity. Plants used in the experiments were grown in a hydro-
ponic system (Srivastava et al., 2009). The substrate used to stabi-
including synthetic chemicals such as b-aminobutyric acid, iso- lize the plants was rock wool placed in a plastic net pot that was
suspended directly in the nutrient solution. An air pump supplied
nicotinic acid (INA), benzol [1,2,3] thiadiazol-7-carbothioic acid-S- air to an air stone that aerated the nutrient solution and supplied
methyl ester (BTH) and acibenzolar-S-methyl (ASM, a derivative of oxygen to the roots of the plants. At the seedling stage, plants were
BTH). Salicylic acid and phosphates have also been shown to be transferred to a continuously aerated 1 L hydroponic container (1 L
activators of the SAR response in a variety of hostepathogen NalgeneÒ large polypropylene amber wide-mouth bottles). Each
systems (Cohen, 2001; Oostendorp et al., 2001; Bokshi et al., 2003; container contained a single plant that was supplied with half-
Edreva, 2004; Vallad and Goodman, 2004). The synthetic strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950).
compound probenazole induces SAR, and has been used to control A 2009 P. pachyrhizi isolate from Quincy, Gadsden County, Florida
rice blast (Magnaporthe grisea) and bacterial blight (Xanthomonas was maintained on soybean plants in the greenhouse as a source of
oryzae pv. oryzae) (Watanabe et al., 1979). Probenazole and its inoculum, and urediniospores were collected fresh from leaves of
active metabolite 1,2-benzisothiazole-1,1-dioxide induce SAR in those plants during the days preceding inoculation and were stored
Arabidopsis by stimulating a site upstream of the point of accu- at 4 C until used.
mulation of SA in the SAR-signaling pathway (Yoshioka et al., 2001).
Probenazole enhances some of the resistance mechanisms associ- 2.2. Saccharin applications to leaves or roots
ated with an oxidative burst that occurs after infection by the rice
blast agent (Gozzo, 2003). It also induces the accumulation of Saccharin (MP Biomedicals, LLC) was applied as either a root
unsaturated fatty acids that act as anticonidial factors (Kessmann drench or sprayed onto the foliage. For the root drench, each plant
et al., 1994). Probenazole has been reported to function upstream received either 30 ml of 3 mM saccharin in deionized water or
of SA in Arabidopsis (Iwai et al., 2007) and tobacco (Lyon, 2007). 30 ml deionized water (as the control) in 1 L half-strength Hoag-
Saccharin (C7H5NO3S) is a metabolite of probenazole (Uchiyama land’s solution. For the foliar application, at V3 (2nd trifoliate fully
et al., 1973) that is able to induce SAR in rice to improve control expanded leaf) and R1 (first bloom) stage (Fehr et al., 1971) the first
of Magnaporthe grisea and Xanthomonas oryzae, possibly via the fully expanded trifoliate leaf of each plant was treated with 3 mM
induction of host defenses, since saccharin has been shown to saccharin or deionized water (for control) using a spray bottle
induce SAR (Oostendorp et al., 2001; Siegrist et al., 1997). (Fisher brand adjustable-spray wash bottle) until runoff occurred.
After addition of saccharin to the nutrient medium, nutrient solu-
Previous observations have highlighted the potential of tion lost via transpiration and uptake was replenished by frequent
saccharin to activate SAR against tobacco mosaic virus (TMV) in additions of half-strength Hoagland’s solution to maintain
tobacco, Colletotrichum lagenarium in cucumber, Uromyces appen- a constant volume.
diculatus in bean (Siegrist et al., 1998), U. fabae in Vicia faba (Boyle
and Walters, 2005) and to the powdery mildew Blumeria graminis 2.3. Inoculation with P. pachyrhizi
f. sp. hordei in barley (Boyle and Walters, 2006) by triggering
signaling at a point upstream of SA accumulation. A large number of Plants were challenged at 1, 5, 10 and 15 d after treatment (DAT)
mechanisms are involved in biocontrol and can involve direct with saccharin. A urediniospore suspension (8000 spores/ml
antagonism via production of antibiotics, siderophores, HCN, deionized water) was applied to the leaves using a hand sprayer
hydrolytic enzymes (chitinases, proteases, lipases, etc.), or indirect (PORTER-CABLE 6-Gallon Air Compressor - Model #: C2002-WK,
mechanisms in which the biocontrol organisms act in a probiotic Porter-Cable Corporation Jackson, TN). Inoculated plants were
fashion by competing with the pathogen for infection and nutrient placed in the vicinity of a humidifier for 24 h to promote ure-
sites. Biocontrol can also be mediated by activation of SAR by diniospore germination. Plants were harvested and assessed for
modification of hormonal levels (Bowen and Rovira, 1999; Van visible soybean rust symptoms 2 weeks after inoculation.
Loon, 2007) in the plant tissues.
2.4. Assessment of the induced disease resistance
Soybean rust (SBR), caused by Phakopsora pachyrhizi Syd. &
P. Syd., is a serious disease of legumes that can cause devastating Two weeks after inoculation, plants were assessed for disease
yield losses in soybean (Glycine max (L.) Merr). It was first reported severity (DS) using a 1e9 scale based on the series of photos in the
in the Eastern Hemisphere more than a century ago (Ogle et al., “Asian Soybean Rust Disease Severity Evaluation Scale” developed
1979). In recent decades, the disease has spread to Hawaii by Bayer CropScience (Bayer CropScience, Research Triangle Park,
(Killgore and Heu, 1994), Africa (Kawuki et al., 2003), South NC). A rating of 1 was assigned to plants showing no visible
America (Yorinori et al., 2002) and North America (Schneider et al., symptoms and a rating of 9 indicated 67.5e100% disease severity.
2005). P. pachyrhizi differs from most other rust fungi in having Sporulation was also rated using a 9-point scale in which a rating of
a large number of legume hosts (Slaminko et al., 2008). 1 was assigned to plants showing no sporulation and a rating of 9
indicated that 90e100% uredinia were sporulating.
The objective of this study was to examine the effectiveness of
saccharin as an inducer of SAR against P. pachyrhizi in soybean
plants. The study also examined the effects of saccharin and
saccharin-induced SAR on plant growth.

2. Materials and methods

2.1. Plant materials 2.5. Biomass analysis

The soybean (Glycine max) cultivars Williams 82 and Benning For growth measurements, plant heights were measured and
were used. Williams 82 is a maturity group (MG) III cultivar from numbers of fully matured leaves were counted. Fresh weights of
the Midwest (Bernard and Cremeens, 1988), and Benning is an MG shoots and roots were determined immediately after harvest, and

728 P. Srivastava et al. / Crop Protection 30 (2011) 726e732

thereafter shoots and roots were dried in an oven at 60 C for 48 h
before dry weights were determined.

2.6. Statistical analyses

The experiment was set up as a completely randomized factorial
design comprised of 3 treatments  2 genotypes  2 plant devel-
opmental stages  4 DAT treatments and each treatment was
replicated three times. The GLIMMIX SAS 9.2 statistical program
(SAS Institute Inc., Cary, NC) was used for data analysis. The control
data from root drench and foliar spray was pooled as there was no
significant difference between the controls.

3. Results

3.1. Disease reactions Fig. 1. Effect of treatment in inducing SAR against the soybean rust pathogen in
soybean plants at V3 or R1 stage treated with saccharin and challenged later with
The application of saccharin before inoculation with Phakopsora pachyrhizi. Error bars indicate standard errors.
P. pachyrhizi reduced disease severity compared to the control
treatment (Table 1). Leaf position and mode of saccharin applica- Saccharin administered as a root drench at the V3 (5.0 DS) and
tion were both significant factors in the severity of rust infection. R1 (4.2 DS) stages was also slightly more effective than the foliar
Saccharin administered as a root drench at the V3 (4.8 DS) and spray at V3 (5.3 DS) and R1 (5.2 DS) in reducing sporulation (Fig. 4).
R1 (6.3 DS) developmental stages was apparently more effective at Decreased sporulation was observed 15 d after the application of
inducing SAR than the foliar spray at V3 (5.4 DS) and R1 (7.5 DS) saccharin as a root drench at V3 (4.6 DS) and R1 (4.1 DS) (Fig. 5). In
(Fig. 1). Increased resistance was observed 1 d after application of contrast to the root drench, foliar treatment with saccharin at V3
saccharin as a root drench at V3 (4.5 DS) and R1 (6.5 DS) (Fig. 2). and R1 plants did not significantly reduce sporulation.
However, systemic protection against rust infection was still
apparent 15 d after application at the V3 (5.3 DS) and R1 (6.0 DS) 3.2. Time-course response
stages, particularly in plants that received the root drench appli-
cation (Fig. 2). In contrast to the root drench treatment, foliar For the time-course response, when plants were challenged with
application of saccharin solution at the R1 stage did not signifi- the pathogen 5 d, 10 d and 15 d after treatment with saccharin at the
cantly induce SAR protection until 15 d after treatment (6.1 DS). V3 stage, the root drench application was associated with signifi-
cant resistance to the disease, whereas the foliar application treat-
The root drench was more effective at inducing SAR than the ment was significant only at 15 d (Fig. 2). The root drench
foliar spray, when the plants were at the R1 stage, with the application was associated with significant resistance to the disease
exception of plants that were inoculated 5 d after either treatment at 1, 10 and 15 d after exposure of saccharin, whereas the effect of
(Fig. 2). However, at the V3 stage, application of saccharin as either the foliar application was insignificant. Increased resistance was
a root drench or a foliar spray was effective in reducing SBR severity observed 1 d after root application of saccharin at the R1 stage, and
(6.7 DS for V3 and 7.8 DS for R1) (Fig. 1, Table 1). was still apparent 15 d later (Fig. 2). In contrast, the foliar treatment
with saccharin at the R1 stage did not increase systemic protection
Application of saccharin at the V3 stage instead of at the R1 until 15 d after treatment. When plants treated at the V3 stage were
stage was more effective in reducing disease severity on all inoc- challenged with the pathogen at 1, 5, 10 and 15 d after the saccharin
ulation dates (Fig. 3). For both genotypes, the saccharin root drench treatment, SAR was induced by 5 d and continued for at least 15 d. In
was more effective than the leaf treatment at most of the time plants inoculated at the R1 stage, SAR was induced within one day
points when rust severity was assessed (Fig. 2). and seemed to be consistent up to 15 d after treatment. Sporulation
was also significantly reduced by the root drench treatment (Fig. 5).
Table 1
Results of analysis of variance of the effect of saccharin on disease symptoms of 3.3. Biomass
soybean leaves challenged with the soybean rust pathogen. (DAT¼days
after treatment, or time between saccharin application and challenge with When SAR was induced by application of saccharin, either to the
Phakopsora pachyrhizi.) leaf or as a root drench, there were no reductions in plant growth
parameters considered (biomass, height and leaves), regardless of
Effect Rust Severity how saccharin was applied to plants except for a reduction in
height and shoot dry weight when saccharin was applied as
DAT *** a drench in R1 and challenged with the pathogen 15 d after
Genotype * treatment.
DAT*Genotype NS
Treatment *** For plants that received the root drench application, there was
DAT*Treatment ** an increase in height, number of leaves, shoot dry weight (SDW)
Genotype*Treatment * and root dry weight (RDW) in the time-course response for all three
DAT*Genotype*Treatment NS treatments except for height and shoot dry weight of plants treated
Stage *** at R1 stage and inoculated 15 d after the root drench application.
DAT*Stage *** These data suggest that the saccharin applications had little or no
Genotype*Stage * effect on plant growth.
DAT*Genotype*Stage NS
Stage*Treatment **
DAT*Stage*Treatment *
Genoty*Stage*Treatment NS
DAT*Genotype*Stage*Treatment NS

Levels of significance (***, P < 0.001; **, P < 0.01; P < 0.05; NS ¼ not signifi-
cant) were determined using GLIMMIX.

P. Srivastava et al. / Crop Protection 30 (2011) 726e732 729

Fig. 2. Time-course responses for protection efficiency of saccharin applications against soybean rust when challenged at 1, 5, 10 and 15 d after treatment (DAT). (Means of three
replications, standard errors indicated.)

Fig. 3. Growth stage effect on efficacy of saccharin in inducing SAR in soybean plants Fig. 4. Effect of saccharin applications on sporulation from uredinia on soybean plants
subsequently inoculated with Phakopsora pachyrhizi urediniospore suspensions. Error treated at the V3 or R1 stages and subsequently challenged with Phakopsora pachyrhizi.
bars indicate standard errors. (DAT¼Days after treatment.) Error bars indicates standard errors.

730 P. Srivastava et al. / Crop Protection 30 (2011) 726e732

Fig. 5. Effect of saccharin application on sporulation from uredinia on soybean plants inoculated with Phakopsora pachyrhizi uredinospores. Days after treatment (DAT) refers to the
time elapsed between saccharin treatment and challenge with Phakopsora pachyrhizi. Error bars are standard errors.

4. Discussion rust fungus Uromyces viciae fabae, and in barley resistance against
powdery mildew (B. graminis f. sp. hordei).
The results of the present study demonstrated that Williams 82
and Benning plants, which are susceptible to soybean rust, appear When systemic protection was induced by application of
to have developed SAR to P. pachyrhizi in response to saccharin saccharin, either to the leaf or as a drench, there was no significant
application. The method of saccharin application was a significant effect on height or biomass of plants. These data suggest that
factor in the severity of rust infection especially when the appli- induction of systemic resistance to rust infection using saccharin
cation occurred at the R1 stage (Fig. 2). Saccharin applied as a root had no adverse effect on plant growth. This is consistent with
drench was usually more effective than the leaf treatment at previous work showing that application of various agents to induce
inducing protection. A similar response has been observed by resistance in barley was not associated with reductions in plant
others in other plant species (Siegrist et al., 1997; Boyle and growth (Dehne et al., 1984; Steiner et al., 1988; Oerke et al., 1989;
Walters, 2005, 2006). The time-course experiments of resistance Kehlenbeck et al., 1994; Mitchell and Walters, 1995; Boyle and
induction clearly showed that maximum induction was reached Walters, 2005, 2006; Walters et al., 2009).
within 1 d after treatment when saccharin was applied as a root
drench, and that resistance of treated plants was not higher when Earlier studies of soybean plantepathogen interactions showed
they were challenged with the pathogen at a later time point that soybean resistance to fungal diseases were correlated with the
(Fig. 2). These results are comparable to those that Boyle and levels of the antibiotic-like phytoalexin glyceollin (Lozovaya et al.,
Walters (2005, 2006) reported in broad bean-resistance to the 2004a). The pathogen-inducible phenylpropanoid phytoalexins
play a crucial role in plant disease resistance and they have been
found in many plant species, including legumes (Keen et al., 1972;

P. Srivastava et al. / Crop Protection 30 (2011) 726e732 731

Dakora and Phillips, 1996; Dixon, 2001; Lozovaya et al., 2004a,b). transition from juvenile to adult during vegetative growth at the
The importance of glyceollin in providing resistance against Phy- flowering transition or with the onset of senescence. The mecha-
tophthora sojae in soybean has been demonstrated in different nism of controlling developmental transitions may also govern the
studies (Ayers et al., 1976; Albersheim and Valent, 1978; Zahringer expression of resistance (Develey-Riviere and Galiana, 2007).
et al., 1978; Bhattacharyya and Ward, 1985; Ebel and Grisebach,
1988; Graham and Graham, 1991). Glyceollin is also toxic to many 5. Conclusion
other important soybean fungal pathogens (Lygin et al., 2009).
Lignin is also a well-studied plant cell wall aromatic polymer that In conclusion, this work presents the effect of saccharin on
helps to provide a barrier to fungal entry into plant tissues and to soybean rust control and on plant biomass under two different
the diffusion of toxins and enzymes which fungi secrete in advance forms of application and at two different stages of plant develop-
of invasion to prepare or soften the plant cells for colonization ment. A root drench application of saccharin was more effective
(Vance et al., 1980; Bruce and West, 1989; Elfstrand et al., 2002). overall in terms of reducing soybean rust severity. In tissue distant
Synthesis of antimicrobial phytoalexins and reinforcement of cell from the inoculation site, SAR was apparently more effective in
walls (due to synthesis of various wall-bound phenolic compounds) younger plants. While saccharin did not yield a direct measurable
is part of a plant’s innate or basal resistance that helps to prevent or effect on plant growth, its positive role on disease suppression may
impede infection by fungi (Lygin et al., 2009). Lygin et al. (2009) lead to better plant productivity. Further experiments are being
reported that phenolic compounds such as glyceollin, for- conducted to elucidate the mode of action by which saccharin
mononetin, and kaempferol inhibits germination and development application reduces disease in soybean plants challenged with
of P. pachyrhizi urediniospores and indicated that increased soybean rust. Physiological and molecular study of defense
synthesis of glyceollin and lignin could help to improve soybean responses is required to establish whether the effects observed
rust resistance. here represent local and SAR.

The induction of plant defenses following pathogen challenge is Acknowledgments
known to be energy-dependent (Smedegaard-Petersen and
Tolstrup, 1985). It is assumed that resistance induced by use of This work was funded by the USDA-ARS and North Central
exogenous agents is therefore also energy-demanding, but an Soybean Research Program. We thank Dr Mrittunjai Srivastava
adverse effect on plant growth may only become apparent when (NFREC, University of Florida) for useful discussions and Dr
the demand on available resources exceeds supplies (Boyle and Dongyan Wang (University of Florida) for help with statistical
Walters, 2005). SAR is characterized by the increased expression analyses.
of a large number of PR genes, in both local and systemic tissues.
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