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��ࡱ�>��	�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������5@	��0�bjbj�2�2	.��X�Xv�(C�������JJJJ�����I�I�I82J�Kd�k�vK�K"�K�K�K�LNM<KM �������$��R՜���V�L�L�V�V�JJ�K�K%��t�t�t�V�J��K��K��t�V��t,�t�tR�����V��KjK�����U��Ip�����D;�0k�����5s��<V���JJJJV������kM�IP�tUR��S�kMkMkM�����v9�#t^��v9The effect of clubroot (Plasmodiophora brassicae) on glucosinolate levels in broccoli (Brassica olerecea L. ssp. italica cv. Marathon) and pak choi (Brassica rapa ssp.chinensis) seedlings 

Nazrul IslamA,B, Rod Jones C and David GuestA

AFaculty of Agriculture, Food & Natural Resources, The University of Sydney, NSW 2006     Australia. Email: HYPERLINK "mailto:david.guest@sydney.edu.au"david.guest@sydney.edu.au 
BCurrent address:  Lab of Soil Microbiology, Dept of Soil Science, University of Saskatchewan, Saskatoon, Canada,  Email: mdi985@mail.usask.ca
CFuture Farming Systems Research, Department of Primary Industries, Knoxfield Centre,
Private Bag 15 Ferntree Gully DC, Vic, 3156, Australia. Email: rod.jones@dpi.vic.gov.au

Corresponding Author: Nazrul Islam, Lab of Soil Microbiology, Dept of Soil Science, College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon, 51, Campus Drive, S7N 5A8, Canada,  Email: mdi985@mail.usask.ca
  	
Abstract 
Brassicas are a rich source of glucosinolates, one of which, glucoraphanin produces the isothiocyanate sulforaphane which has been shown to possess chemo-protective activity in humans. Glucosinolates are thought to help defend plants against herbivore and pathogen attack. Clubroot, caused by Plasmodiophora brassicae, is an important and common disease worldwide, however the effect of clubroot on glucosinolate levels is unclear. The combination of incorporation of infected gall slurry into potting mix before planting with a post-planting application of spore suspensions to the root zone was found to cause faster and more consistent disease development than single inoculation methods. Clubroot infection significantly affected glucosinolate levels in both field and glasshouse grown brassicas. In a glasshouse trial, P. brassicae affected glucosinolate levels in both root and aerial tissues of broccoli (Brassica olerecea L. ssp. italica cv. Marathon) seedlings during primary, secondary and mature gall formation. Aliphatic glucosinolate levels (glucoiberin, progoitrin, glucoraphanin, gluconapin) remained constant in aerial tissues but significantly increased (1.5 to 2.0 times) in root tissues during symptom development (28 days post inoculation). Indole glucosinolates (4-hydroxy glucobrassicin, glucobrassicin and neoglucobrassicin) in root tissues increased 2.5 fold during symptom development to mature gall formation stage (28 to 42 days) and also significantly increased (P<0.05) in aerial tissues (1.25 to 2 fold). Glucobrassicin levels in root tissues increased 8-fold during symptom development. Field infections of Brassica rapa ssp.chinensis seedlings with clubroot caused decreased progoitrin levels in leaf and stem tissues, but significantly increased in roots. Indole glucosinolate levels also increased significantly in infected roots, being up to three times higher in infected root tissues. These results indicate that glucosinolate classes were affected differently in varying plant tissues upon clubroot infection.

Keywords: glucosinolate, clubroot, brassica
 Introduction

Plasmodiophora brassicae is one of the most damaging soil-borne pathogens of the Brassicaceae, causing clubroot disease worldwide (Woronin 1878; Ludwig-M�ller et al. 1999a & 1999b). Infection of roots may occur at any stage of plant growth although typical clubroot symptoms are not obvious until the final stages of disease development. Root galls interfere with the normal uptake of water and nutrients, leaving plants stunted and prone to wilting. 

Gall development resulted from abnormal tissue proliferation caused by an interference with auxin (Butcher et al. 1976; Kavanagh & Williams 1981) and cytokinin (M�ller & Hilgenberg 1986) metabolism. In the Brassicaceae auxin is synthesised via the indole glucosinolate pathway (Mahadevan & Stowe 1972; Ludwig-M�ller et al. 1993), and there is evidence that clubroot infection increased levels of indole glucosinolates and auxin precursors in aboveground tissues of B. campestris (syn. Brassica rapa L. subsp. campestris (L.) and B. oleracea (Anonymous 2009; Ludwig-M�ller et al. 1999b). 

Over 120 glucosinolates have been identified, mainly in species belonging to the Brassicaceae (Fahey et al. 2001). Glucosinolates impart quality and flavour characteristics to brassica vegetables, as well as resistance against non-adapted pathogens and herbivores (Mithen 2001; Bennett & Wallsgrove 2006). Epidemiological and in vitro studies have recently indicated that diet and some cancers are closely interlinked, and natural phytochemicals, particularly isothiocyanates derived from specific glucosinolates, such as sulforaphane from glucoraphanin, have protective activity particularly during the initiation and promotion phases of human cancer development (Talalay & Fahey 2001; Farnham et al. 2004; Anilakumar et al. 2006). 

The glucosinolate content of brassica tissues is influenced by genetics, environmental factors, tissue type and the stage of development (Jones et al. 2007). Furthermore, accumulation of glucosinolates can be induced after wounding and pathogen attack (Koritsas et al. 1991; Bodnaryk 1992).  The effect of plant pathogens such as P. brassicae on glucosinolate accumulation in above-ground and below-ground organs of brassica crops is relatively unknown. The main objective of this study was to monitor the accumulation pattern of aliphatic and indole glucosinolates in both above- and below-ground tissues of Brassica oleracea ssp. italica cv. Marathon (broccoli) and Brassica rapa ssp. chinensis (Chinese cabbage) as affected by clubroot diseases development in glasshouse and field trials. It was hypothesized that accumulation rate of alipathic and indole glucosinolates in shoot and root tissues was greatly affected with the sequential development of primary and secondary phases of the pathogen, P. brassicae during root infection (gall formation) process.  This information could be useful to explore the pathway of the glucosinolates synthesis for the phytomedicinal manipulation of glucosinolate levels in brassicas.


Materials and Methods
Field samples

Whole plant samples were collected from healthy and Plasmodiophora brassicae-infected five week old Brassica. rapa ssp. chinensis cv. Sumo at Camden, NSW, Australia in March 2008.  Four replicated samples of above and belowground parts of plants were kept in airtight polyethylene bags with moist field soil for 3 hours during transport back to the laboratory and thorough washing and drying between filter paper took place. Washed, dry samples were immersed into liquid nitrogen and stored at -200C.

Glasshouse trial: Broccoli 

Broccoli (Brassica oleracea L. var. italica) cv Marathon (Fairbanks Seeds, West Melbourne 3003, Australia) was used as a susceptible host. Plastic pots (1.4L) were filled with potting mix consisting of coconut peat and washed sand (1:1 w/v) supplemented with 10 g kg-1 Osmocote slow release fertilizer (Scotts Australia Pty. Ltd). Seedlings were grown in a glasshouse maintained at 22 to 25�C during day and 18 to 20�C at night, with an RH of 80 to 90%. Seedlings were irrigated three times daily for 3 minutes each. Pots were thoroughly watered immediately prior to inoculation and seed sowing. Seedlings were harvested 7, 28 and 42 days after inoculation, and divided into aerial and root parts and immediately frozen in liquid N2 for glucosinolate analysis. 

Clubroot inoculum

The inoculum consisted of either a field isolate of Plasmodiophora brassicae Woronin of broccoli clubroot sample collected by Dr. Caroline Donald (DPI Victoria) from a broccoli farm in Werribee South, Victoria, Australia in 2006 used in the broccoli trial, or fresh clubroot galls on bok choy (Brassia rapa L. var. chinensis), collected from Camden NSW in March 2008. Galls were washed and stored in a freezer at -20�C until use. 
Frozen root galls were homogenised with three volumes of distilled water (w/v) in a blender, filtered through nylon stocking, then washed by repeated centrifugation at 2000 g for five minutes and resuspension of the pellet in sterile distilled water (Donald and Porter 2004). Spore viability was estimated by vital staining with 0.01% (w/v) hypertonic neutral red in 10�mM phosphate buffer (pH 7.5) (O�Connell et al. 1985), and spores were diluted to the required concentration with water. Inoculation involved mixing 50 mL/kg inoculum slurry (107 spores/mL) with the potting mix 3 days before transplanting. After transplant a further 200 �L of spore suspension (108 spores/mL) was applied to the base of each seedling (Donald & Porter 2004; Toxopeus & Janssen 1975).

Glucosinolate analysis

Plant samples were freeze dried, ground with a coffee blender to a fine powder and stored in air-tight containers at room temperature until HPLC analysis (Jones et al. 2007). Powdered samples (0.4 g) were preheated for 10 minutes in a water-bath (900C) in 10 mL centrifuge tubes, then 10 mL of boiling filtered, deionised water was added and the tubes heated for a further 10 minutes. Samples were centrifuged at 4,000 rpm for 10 minutes. The supernatant was collected and transferred to a 25 mL volumetric flask. The pellet was re-suspended using a vortex mixer with 10 mL of filtered, deionised boiling water and re-centrifuged at same speed. The supernatant was combined and the volume adjusted to 25 mL. The extracted supernatants were filtered (Phenex NY, 0.45 �m regenerated cellulose) into auto-sampler vials (2 mL AMB RAM vials, 9 MM THD) for High Performance Liquid Chromatography (HPLC) analysis (Jones et al. 2007). 

 Glucosinolates were separated from 20 �L samples injected onto a C-18 column as described by West et al. (2002). Column temperature was maintained at 300C and column pressure at 11.5 MPa. The column was eluted with a binary gradient of 50 mmol/L ammonium acetate in pure water at pH 6.74 (mobile phase A) and 20% methanol in 50 mmol/L ammonium acetate (v/v) (mobile phase B) at a flow rate of 1 mL/min. Gradient was maintained at 0% (mobile phase B), for 10 minutes, then increased to 40% (mobile phase B) over 5 min, maintained at 100% (mobile phase B) for 10 minutes before re-equilibrating the column with the 100% (mobile phase A) for 15 min. Absorbance was monitored at 230 nm. Individual glucosinolates were identified by comparison with purified standards: glucoiberin (3-methylsulphinyl-propyl), progoitrin (2OH-3-butenyl), glucoraphanin (4-methylsulphinyl butyl), gluconapin (-3butenyl), 4-hydroxyglucobrassicin (4OH-3-indolylmethyl), glucobrassicin (Indol 3 ylmethyl), neoglucobrassicin (1-methoxyindol-3-ylmethyl). Standards were purchased from C2 Bioengineering (www.glucosinolates.com), Hovedgaden 12, 2690 Karlslunde, Denmark.


Results
Clubroot infection 

The sequence and timing of infection and symptom development was monitored in glasshouse-grown and inoculated seedlings. Primary plasmodia infected and differentiated in root hairs to form zoosporangia within 7 days of inoculation (Figure 1A-D). Secondary plasmodia were first observed in cortical cells 7 days after inoculation, and proliferated to develop extensive multinucleate plasmodium (Figure 1E-G), causing host  cortical cell destruction (Figure 1H)  by 10 days. Visible galls also appeared 10 -12 days after inoculation (Figure 1I-J), and continued to grow. Resting spores were first seen 21 days after inoculation, and matured after 42 days (Figure 1K & L). Resting spore maturation confirmed treating 0.01% (w/v) hypertonic neutral red in 10�mM phosphate buffer (pH 7.5), Majority of resting spores absorbed reddish color and few of spores did not have colored remain whitish seen under microscope (data not show, Figure 1L).

Effect of clubroot infection on glucosinolate levels in broccoli seedlings

The major glucosinolates measured in the aerial parts of broccoli seedlings were (in order): glucoraphanin, glucobrassicin, glucoiberin,  and neoglucobrassicin (Fig-2, data not shown). Inoculation with clubroot caused significant changes in individual glucosinolate contents in broccoli seedlings (Tables 1 & 2), but results varied between glucosinolate types and tissues. Tables 1 & 2 portray the differences in glucosinolate contents between control and inoculated seedlings, hence values are not an accurate indicator of actual glucosinolate content. In aerial tissues (stems and leaves) no significant changes were recorded in the aromatic and aliphatic glucosinolates: glucoiberin, progoitrin, glucoraphanin or gluconapin content between 7 and 42 days after inoculation (Table 1). Significant increases were observed, however, in the indole glucosinolates 4-hydroxy glucobrassicin (at Days 7, 28 and 42), glucobrassicin (Days 7 and 28) and neoglucobrassicin at Day 42. No discernable pattern was seen in changes in indole glucosinolates in aerial tissues after clubroot infection. 4-hydroxy glucobrassicin increased for the duration of the experiment; glucobrassicin declined after 28 days and neoglucobrassicin only increased at Day 42.

A very different pattern in glucosinolate degradation or accumulation was observed in broccoli root tissues (Table 2). Major glucosinolates were: glucobrassicin, neoglucobrassicin and glucoraphanin (Fig-2, data not shown). Significant declines were observed on day 7 in glucoiberin, glucobrassicin and neoglucobrassicin in root tissue growing in inoculated media, while all glucosinolates increased significantly on day 28 (Table 2). By day 42, only the indoles 4-hydroxyglucobrassicin and glucobrassicin levels were significantly higher in seedlings growing in inoculated media. 

Effect of clubroot on glucosinolate contents in field-grown Chinese cabbage (Pak-Choy)

The major glucosinolate in both shoots and roots of 5 week-old field grown Pak Choy was progoitrin, with lesser amounts of glucoraphanin and gluconapin (Tables 3 & 4). Clubroot infection significantly reduced progoitrin content in aerial tissues of diseased plants, but increased gluconapin, glucobrassicin and neoglucobrassicin levels (Table 3). Progoitrin levels were 18% lower in infected plants (P=0.013), although levels of the minor aliphatic glucosinolate, gluconapin, increased (P=0.03). There were no significant differences in glucoraphanin levels. Glucobrassicin was not detected in healthy shoots, whereas infected shoots produced 0.44 �mole/g DW. 

All measured glucosinolates, except gluconapin, increased in clubroot-affected root tissues (Table 4). Progoitrin content increased by nearly 4-fold. The glucoraphanin content of roots was very low, but significantly higher (P<0.001) in diseased roots. Gluconapin levels were low in healthy roots, and significantly lower (P<0.001) in diseased roots. Clubroot infection significantly increased indole glucosinolate levels of Pak Choy roots (Table 4). Diseased roots had significantly higher levels of 4-hydoxyglucosinolate, glucobrassicin (P<0.01) and neoglucobrassicin (P<0.01). 

Discussion
Inoculation Method
Rapid and reproducible clubroot symptoms developed after transplanting broccoli seedlings into pots containing slurry of infected roots followed by a post-transplant soil drenching with P. brassicae spores. Root hair infection, indicated by the appearance of primary plasmodia and zoosporangia in infected root hairs, was seen within 7 days of transplanting, followed by the development of multinucleate secondary plasmodia in the root cortex and host cell destruction by 10 days (Fig. 1). The first visible galls appeared after 10-12 days and resting spores developed by 21 days and matured after 42 days. While Dekhuijzen (1979) reported amoeboid structures within cortical cells after 10 days, visible galls were not seen until 14 days after inoculating B. rapa with resting spores of P. brassicae. The presence of host factors in decaying root tissue used as a slurry inoculum may stimulate the germination of resting spores (Williams & McNabola 1967; Macfarlane 1970). Root hairs on 5�6 day old roots are reported to be more susceptible to infection by P.  brassicae than younger or older root hairs (Samuel & Garrett 1945; Naiki et al. 1978; Asano et al. 2000). In the present study inoculum slurry was added to the potting mix 3 days before transplanting and spores were added at transplanting, so that root hairs were exposed to the pathogen as they developed, resulting in rapid infection. A double inoculation technique ( potting mix amended with inocula slurry and extracted spores injected into root zone of transplanted seedlings) has been used for P. brassicae, and the rapid and consistent formation of galls within 10-12 days makes it a useful method for future studies. Single and double inoculation technique were  evaluated examining  percent root hair, cortical cell infection and gall size found faster and rapid infection and bigger gall size by double inoculation than any of single inoculation methods used in this study (Data not shown).

Effect of Clubroot Infection on Glucosinolate Content

Glucosinolates and their resultant isothiocyanates are believed to play a major role in host-pathogen interaction. There is evidence for the association of glucosinolates and their breakdown products in resistance to fungal pathogens and insects (Mithen 1992; Ludwig-M�ller et al.1999a & 1999b; Rangkadilok et al. 2002). The main objective of this study was to monitor changes in the levels of individual glucosinolates in the aerial tissues and roots of broccoli and pak choi seedlings following P. brassicae inoculation and subsequent symptom development.  The major glucosinolates measured in the aerial parts of broccoli seedlings were (in order): glucoraphanin, glucobrassicin, glucoiberin and neoglucobrassicin (data not shown), in agreement with Kushad et al. (1999).  The major glucosinolate in both shoots and roots of 5 week-old field grown pak choy was progoitrin, with lesser amounts of gluconapin and glucoraphanin (Tables 3 & 4). This is in partial agreement with He et al. (2000) who found pak choi (Brassica campestris) contained gluconapin, progoitrin, glucobrassicin and neoglucobrassicin, but no glucoraphanin. 

Glucosinolate levels in Brassica aerial tissues and roots responded in different ways to P. brassicae infection of the roots. While no significant changes were recorded in aliphatic and aromatic glucosinolates in aerial tissues in broccoli seedlings, indole glucosinolates (4-hydroxyglucobrassicin, glucobrassicin, and neoglucobrassicin) all increased significantly in both aerial and root tissues (Tables 1 & 2). In 5 week old pak choi seedlings, however, a different pattern was observed (Table 3). The dominant glucosinolate, progoitrin, declined significantly in aerial tissues, while gluconapin, glucobrassicin and neoglucobrassicin all increased significantly (Table 3). It appears that clubroot infection can therefore both induce and decrease glucosinolate content, but the reason for this differential response is not known.

Compared to healthy broccoli seedling roots, levels of  4-hydroxyglucobrassicin and glucobrassicin in inoculated roots decreased significantly during primary root hair infection (Day 7; Table 2) but increased dramatically during gall formation (Day 28; Table 2).  In pak choi, however, all glucosinolates increased significantly in infected roots compared to healthy controls when measured 5 weeks (35 days) after transplanting (Table 4). Despite a significant decline in progoitrin in infected aerial tissues, progoitrin increased dramatically in infected pak choi roots (Table 4), again indicating different patterns in glucosinolate synthesis and metabolism in plant parts upon clubroot infection.
 
Doughty et al. (1991) and Rostas et al. (2003) reported higher levels of glucobrassicin, neoglucobrassicin and 4-methoxy�glucobrassicin in rapeseed leaves infected with Alternaria brassicae, however there have been few other published studies on systemic effects of clubroot on glucosinolates. P. brassicae infected Tropaeolum majus leaves contained more than 150% higher levels of the benzylglucosinolates glucotropaeolin, gluconasturtiin and glucomalcomiin than leaves from uninoculated plants (Ludwig-M�ller et al. 1999b; Ludwig-M�ller & Cohen 2002). The same study reported that benzylglucosinolate levels of young Carica papaya leaves decreased by 29% following infection, while levels in old leaves increased by 7%. 

Transportation of glucosinolates via the phloem over long distances (Chen et al. 2001) is not likely to be the main cause of the observed differences in root and shoot patterns, and the most plausible physiological explanation is that glucosinolate biosynthesis and turnover is regulated differently in each organ. Several glucosinolate transcription factors show organ specific expression patterns (Gigolashvili et al. 2008). Devos et al. (2006) showed that myrosinase was up-regulated during the early stages of infection and Siemens et al. (2006) reported the down-regulation of myrosinase in Arabidopsis thaliana during the later stages of gall formation. Myrosinase converts indole glucosinolates to their corresponding nitriles and then to indole acetic acid (IAA) (Fenwick et al. 1983), showing that indole glucosinolates can be alternative precursors of auxin production. The formation of auxin from indole glucosinolates following infection may then induce clubroot symptoms (Mithen 1992).  There are several factors that still need to be investigated before a precise role can be attributed, including changes in tryptophane oxidase activity after infection, the activity of nitrilase and the pH of infected cells, and how this influences the products from myrosinase-catalysed degradation (Ludwig-M�ller et al. 1999a & 1999b).. Butcher et al. (1974) proposed that indole glucosinolates are converted by clubroot to indole acidic acid (IAA) during clubroot formation, and vegetative secondary plasmodia of the pathogen are known to produce cytokinins (M�ller & Hilgenberg 1986), the increased amount of IAA is more likely to result from the increased synthesis and turnover of auxin precursors in infected roots (Rausch et al. 1983). However, a more detailed understanding of tissue specific regulation of glucosinolate synthesis and turnover is needed to elucidate the role of specific genes
There is little published information on the response of aliphatic glucosinolates during clubroot development, as previous studies focused on indole glucosinolates (Agerbirk et al. 2008). Our results confirm that glucoraphanin is the major aliphatic glucosinolate in broccoli roots and shoots, and that shoots contain more aliphatic glucosinolates than roots (Kushad et al. 1999). Aliphatic glucosinolate levels in infected broccoli and pak choi shoots fluctuated as galls and resting spores developed, but there were no significant differences recorded other than gluconapin in pak choi (Tables 1 & 3). Levels in broccoli roots, however, significantly increased at Day 28, before declining (Table 2). Previous studies have also shown variable responses during clubroot development. Haughn et al. (1991) reported that total aliphatic glucosinolate content in Arabidopsis thaliana during gall formation increased slightly. Application of induction hormone (jasmonic acid) to leaves of Arabidopsis thaliana Col-0,   alipathic glucosinolate increased 1-4 fold (Mikkelsen et�al. 2003) however, Kliebenstein et�al. (2002) reported that alipathic glucosinolates levels following jasmonate induction in leaves became ups and downs.
Clubroot-infected roots of the Chinese cabbage cvs �Granat� and �Osiris� accumulated higher levels of aliphatic glucosinolates during the whole infection period (Ludwig-M�ller et al. 1997), similar to our results where aliphatic glucosinolates increased 28 days after infection (Table 2). 

Given that it is postulated that glucosinolates, and their resultant isothiocyanates, act to protect plants from pathogen and herbivore attack (Bennett & Wallsgrove 2006), it is interesting to speculate on the different responses seen in specific glucosinolate synthesis or metabolism in aerial and root tissues in the present study. Ludwig-M�ller et al. (1997) reported that the total indole glucosinolate content of susceptible cabbage seedlings increased as clubroot symptoms developed, while levels in a resistant cultivar did not, however their work did not distinguish individual indole moieties. In contrast Mullin et al. (1980) were not able to correlate changes in indole content with clubroot formation in susceptible cabbage seedlings, but again roots were not studied. In the present study, all measured glucosinolates in broccoli root tissue increased significantly by 28 days after infection (Table 2), indicating a general response to infection by all glucosinolate types, with marked increases in indole glucosinolates, particularly glucobrassicin. This observation was mirrored in pak choi roots (Table 4), however in this instance progoitrin content increased markedly, with relatively minor increases in indoles. In the aerial tissues of broccoli seedlings only indole glucosinolate synthesis was induced, while in pak choi, progoitrin appeared to be metabolized, or biosynthesis inhibited, while slight increases in production of gluconapin and the indole glucosinolates were observed (Table 3). It is generally considered that there are three classes of glucosinolates found within brassicas: aromatic (derived from phenylalanine), aliphatic and alkenyl (derived from methionine) and indole, or indolyl, derived from tryptophan (Wallsgrove & Bennett, 1995). It is therefore plausible, based on our results that clubroot infection induced indole glucosinolate production from tryptophan in broccoli and pak choi roots, and progoitrin production from methionine in pak choi roots. 

Acknowledgments
The first author�s PostGrad studies (2007 to 2008) at The University of Sydney were supported by Government of Australia (AusAID scholarship). We thank Dr. Caroline Donald and Michael Imsic, Department of Primary Industries, Knoxfield (Victoria, Australia), Len Tessoriero, I&I (NSW, Australia) and Professor Jutta Ludwig-M�ller, Botanisches Institut, Dresden (Germany) for valuable technical assistance and advice during study period.

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