An insight into the basis of resistance in Sorghum bicolor against Colletotrichum sublineolum

This study was done to investigate biochemical, histological and molecular basis of resistance after a compatible and incompatible interaction between Sorghum varieties and Colletotrichum sublineolum. In the susceptible variety, the pathogen invaded all types of tissues but in resistance variety, pathogen invasion was redistricted. Localized cell death was obvious in cortical region of resistant variety that stopped further invasion of pathogen. Epidermis of susceptible variety ruptured shortly after pathogen inoculation and fungal bodies emerged whereas in resistant variety a long delay was noted in occurrence of same event. A steep increase in total phenolics and enzymes related to phenylpropenoid pathway was observed after pathogen challenge in resistant variety as compared to the susceptible one. Histochemical studies revealed heavy deposition of lignin, callose, suberin, phenolics and peroxidases in the resistant variety. These enhanced depositions reduced the pathogen invasion in the host plant. Similarly, RT-PCR analysis revealed higher expression levels of PR protein-genes in the resistant variety. This study presents the nature of a resistant host defense mechanism against a virulent pathogen and made us to integrate defense mechanism for development of resistant varieties in future.


INTRODUCTION
The interactions between plants and pathogens are specific, complex and dynamic.When a pathogen attacks, profound changes in plant metabolism take place.Plants defend themselves from pathogens by several traits that restricts invasion and colonization of the pathogen inside the plant body.These traits are either expressed in plants constitutively as preformed defense related biochemical and barriers or inducible that is formed after pathogen attack.These defenses related traits are expressed not only on site of attack of pathogen but also on non-infested plant parts.Inducible expression of defense related traits in plants was first discovered in the case of fungal and bacterial pathogens in the early nineteen century by researchers (Karban et al., 1999).On a cellular level, ion influx and reactive oxygen species are produced in plants along with the activation of defense related genes.These genes encode proteinase inhibitors (PINs) and pathogen related (PR) proteins *Corresponding author.E-mail: meher_waheed@yahoo.com.(Ebel et al., 1976;Dang et al., 2001;Gtehouse, 2002;Kessler et al., 2002).Plant defense mechanism can be categorized as structural and biochemical mechanism.Structural mechanism restricts the invasion and colonization of pathogen by constructing mechanical barriers.Whereas biochemical mechanism produces substances toxic to pathogen in the plant body (Kombrink and Somissich, 1995;Lebeda at al., 2001).
Plants chemicals that play role in defense systems are mainly secondary metabolites.These secondary metabolites play an important role in interaction of plants with pathogenic microbes.Phenolics are major defense related biochemicals that are synthesized from shikimic acid pathway.These defense related biochemical accumulate in leaves, stems and roots of many plants in response to pathogen attack (Benhamous and Mansfield, 1992;Dixon, 1980).Peroxidases are key enzymes playing role in plant defense system.These enzymes are heme-containing proteins.Function of peroxidase enzyme is to convert hydrogen peroxide into water inside the plant body.But these enzymes can also produce hydrogen peroxide that plays an important role in plant defense response (Apel and Hirt, 2004).Physical defense systems include lignification, suberization and deposition of callose (Hammerschmidt et al., 1985;Stein et al., 1993;Benhamous and Mansfield, 1992).Occlusion of xylem vessels is a common phenomenon caused by microbial invasion or different materials secreted by microbes in xylem vessels (Shah and Babu, 1993).
Colletotrichum is a large genus of ascomycetes.These are among the most successful fungal pathogens causing severe damages to crops worldwide (Bailey et al., 1992).Scientists have carried out extensive studies on different aspects including biology and infection process caused by this pathogenic genus (Bailey et al., 1992).Members of this genus have been used as model for studying infection process, host parasitic interactions and disease development (Perfect et al., 1999).Anthracnose is a destructive disease of Sorghum bicolor (L.) Moench worldwide caused by Colletotrichum sublineolum (P.Henn., Kabát & Bubák).This disease is more common in tropical and subtropical regions with warm and humid climate.Anthracnose infection can be observed on all aboveground parts of the sorghum plant including leaf, stalk, panicles and seeds.
Plant-pathogen interactions are variable in their outcomes and only a few patterns have been identified at present.It is important to understand plant pathogen interactions because these show dynamics of plant defense mechanism.Based on these outcomes, new resistant plant varieties can be bred.During the last years, a few studies have been carried out to know how plant responds to pathogen attack.The present study is a step further in this regards.This article describes anatomical and histochemical responses observed in stalk of sorghum following inoculation with C. sublineolum.

Plant growth and disease development
Preliminary experiments were conducted to screen susceptible and resistant sorghum verities against a virulent strain of C. sublineolum FBL 03.For further experimentation one resistant (Krishna) and one susceptible variety (SR103) was selected.Seedlings of both sorghum verities were raised in plastic pots of 10 inch diameter at rate of three seedlings per pot under sterilized conditions.These seedlings were maintained under aseptic conditions in growth chamber at 25 to 28°C at 80 to 90% of relative humidity.After 30 days of seedling emergence, these were challenged with spore suspension of C. sublineolum prepared in distilled sterilized water.Whereas control were only sprayed with distilled sterilized water.These plants were left for development of disease.
After inoculum application, stems and leaves of both resistant and susceptible plants were taken at different intervals for histological observations.Whole stem and leaf mounts were cleared with saturated solution of chloral hydrate.These cleared mounts were cut into fine sections with microtome and stained with 0.05% Toludine blue followed by fixation in 50% glycerin.To study xylem occlusions, stem mount of 10 cm in length were dipped in acid fuchsine for 30 min.Then these mounts were cut into fine sections with sharp razor blades and observed under microscope.

Biochemical and histological studies
Defense related biochemicals were quantified at regular intervals from starting from day of inoculation.Total phenolics were quantified by using Folin Ciocalteau reagent (Zieslin and Ban-Zaken 1993).Peroxidase (PO) quantification was performed by guaicole method (Fuj, 2001).Polyphenoloxidase were quantified by using catechol as substrate (Mayer and Harel, 1997).Phenylamonialyase were quantified according to methods of Burrell and Rees using L-phenylalanine (Burrell and Rees, 1974).Glucanase were quantified according to method of Simmons (1994) by using leminarin as substrate and chitinase was quantified by using crab shell chitin (Mabuchi, 2000).
For histochemical studies to detect lignin deposition, sections were stained, with Maaule and Wiesnser reagents (Jensen, 1962).Staining with Maule regent was carried out by immersing sections in 1% KMnO4 solution followed by washing with distilled sterilized solution.Then, immersing in 3% HCl solution followed by staining in  25% NH4OH for 1 min and again washing in distilled sterilized water.Maaule reagent stains guaiacyl lignin a dark brown color and syringyl lignin a pinkish red color.For staining with Winser reagent, sections were dipped in acidic solution of 1% phloroglucinol.With this reagent, coniferyl lignin stains purple red color.Suberization was detected by staining fresh fine sections in ethanolic solution of Sudan III for 30 min.Histochemical localization of phenolics was studied by treating fine transverse stems sections in series of alcohol and staining with ferric chloride solution.Peroxidase localization was observed by treating sections in 0.05% hydrogen peroxide and immersing sections in 20 mM Guaicole substrate dissolved in 0.1 M Na acetate buffer at pH 6.0.These sections were then left for incubation in the dark at room temperature.Callose deposition was detected by staining with 0.05% aniline blue and induced fluorescence was observed under microscope.All observations were made under Nikon microscope fitted with digital camera.

RT-PCR analysis
RNA was extracted by using Trizol reagent provided by Invitrogen® according to manufacturer instructions.Relative gene expression analysis was performed according to the method proposed by Ma et al. (2009).Two-step RT-PCR was performed.Single-stranded cDNA was synthesized using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Enzynimics) and oligo-dT primer with total RNA following the manufacturer's instructions.This single stranded cDNAs were used as templates for PCR assays using gene specific primers (Table 1).Amplification was performed in a 10 μl reaction mixture (Enzynomics), 0.5 μl of each primer (10 pm/μl), 3.2 μl of nuclease free water and 1.0 μl of cDNA.The amplification conditions were 94°C for 2 min, followed by 30 cycles of 94°C for 1 min, 50 to 55°C for 1 min, and 72°C for 1 min.Following amplification, a 10 min extension at 72°C was added at the end of the run.In all RT-PCR runs, appropriate negative controls containing no cDNA template were subjected to the same treatment.

Statistical analysis
All the data were analyzed with a one-way analysis of variance (ANOVA) with the help of computer aided program "DASTAT".

Disease development
Our experimental design involved the use of one resistant and one susceptible variety of sorghum and a virulent strain of C. sublineolum.A combination of approaches was used to assess the basis of resistance in sorghum against C. sublineolum.After infestation of both sorghum verities by pathogen, symptoms appeared first on leaves then on the stalk.In resistant variety, numbers of lesion/cm 2 were 73% less as compared to susceptible variety.
To understand cytological basis of resistance in sorghum and C. sublineolum pathosystems, microscopic analysis were performed on both resistant and susceptible verities inoculated with pathogen.Pathogen penetration was found to be associated with different types of modifications in both verities at the cytological levels.These modifications are summarized as follows.Epidermal layer fractured and fungal bodies emerged out on plant surface in susceptible variety (Figure 1a) at 4 days post inoculation (DPI), whereas it remained intact in resistant variety (Figure 1).Pathogen hyphae penetrated all types of tissues including epidermis, cortex xylem and ground tissues in susceptible variety, whereas its invasion was limited to epidermis and cortical region in resistant variety even after 4 DPI.In contrast with the susceptible variety, resistant variety resulted in disintegration of cells in cortical region (Figure 2c) at 2 DPI.It showed a formation of hollow area after clear degradation of the cells as indicated by the microscopic observations of stalk section (Figure 2d).Many xylem vessels were partially occluded with pathogen masses in resistant variety, whereas in susceptible variety, severe occlusions of xylem vessels were observed.Meta xylem vessel lumens were filled completely with fungal hyphae in susceptible variety at 6 DPI.

Biochemical analysis
Calorimetric assays were performed to quantify different defense related biochemicals in sorghum verities with varying susceptibility against C. sublineolum.Figure 3 represents changes in defense related biochemicals at different intervals.There were significant differences in total phenolics in resistant variety as compared to susceptible one.In comparison with susceptible variety, total phenolics quantity was 66, 51, 37 and 73% more in resistant variety at 2, 4, 6 and 8 DPI, respectively.Maximum levels of total phenolics were recorded at fourth DPI of C. sublineolum but started decreasing at 6 DPI (Figure 3).Peroxidase levels were also 23, 34 and 67 higher in resistant variety as compared to susceptible one at 2, 4, 6 and 8 DPI.PO exhibited increasing trend through the experiment in resistant cultivar.Same type of trend was also observed in the case of PPO and PAL activity (Figure 3).Both were in higher quantities in resistant variety.Varying trend was recorded in chitinase activity.It showed less difference in quantities when comparisons were made between resistant and susceptible variety.Seemly, β-1,3-glucanase activity was 34, 68, 51 and 43 higher in resistant variety after attack by pathogen.Highest levels of β-1,3-glucanase were observed at 6 DPI (Figure 3) in resistant variety.

Histochemical analysis
To document histochemical basis of resistance, selective staining were performed for characterizing response of both verities against pathogenesis process and illustrated in Figure 2. Localization of lignin in tissues was observed with phloroglucinol staining of leaves and stalk of both resistant and susceptible verities.Intense lignification was observed in epidermal tissues of leaves (Figure 2b) and stalk (Figure 2a), round xylem vessels and parenchyma cells of stalk at the periphery of vascular tissues (Figure 4f) in resistant variety.Enhanced deposition of both syringyl and guaiacyl lignin was observed after staining with Maaule reagent in stalk of resistant variety (Figure 4f) as compared to susceptible one (Figure 4e).Syringyl lignin mainly constituted endodermal cells in stalk of resistant variety.Guacyl lignin deposition was mostly in sclerenchyma cells of vascular bundles (Figure 4f).
Suberin was deposited in cell walls of endodermal tissues in resistant variety that was visualized as red to brown color after reacting with Sudan III reagent (Figure 5e).This localization was limited up to cell wall but not in whole cell (Figure 5e).This shows that staining reaction was closely associated with localization of suberin.Leaf tissue also exhibited deposition of suberin in resistant   Phenolics deposition was observed in all types of tissues in leaves and stalk of resistant variety.Extensive phenolic deposition was seen in vascular region.Cell walls of xylem were heavily esterified with phenolics (Figure 4d) in only resistant variety.Cells in cortical region and ground tissues also exhibited deposition on phenolics at varying levels.The parenchyma cells of the xylem vessels showed extensive deposition of phenolics in stalk of resistant variety (Figure 4d).In ground tissues, lumen of some cells were completely filled with globules of phenolics (Figure 5f) and at some places, walls of sclarenchymatous tissues were esterified with phenolics (Figure 5g).Phenolics deposition was also observed in the form of small granules present inside sclarenchyma cells.Susceptible variety also exhibited phenolics deposition in vascular region but not as much intense as in diseased plants (Figure 4c).
Peroxidases were stained brown after reaction with guaicol substrate and easily observed under bright field microscopy.Their localization was noted in different types of tissues of both leaves in stalks in resistant variety.Extensive localization of peroxidases was observed in sclerenchyma cells of resistant variety (Figure 5a).Some cell walls of epidermis (Figure 5b) and vascular parenchyma (Figure 5c) were found to be completely esterified with peroxidases in resistant variety.In susceptible variety, deposition of peroxidases was only observed in some sclerenchyma cells.Callose deposition was observed in the form of bright florescence after reaction with aniline blue stain (Figure 5h) in parehchymatous cells of both resistant and susceptible verities but its intensity was more in resistant variety.

RT-PCR analysis
To gain insight into molecular basis of resistance, some defense related genes were analyzed for their expression levels in response to pathogen attack.It was observed that expression levels for all these genes were significantly higher in resistant variety as compared to the susceptible one.Large differences were observed in the expression levels of four genes studied at different intervals after pathogen inoculation.Highest expression level of PR 2 gene was observed at 6 DPI in both resistant and susceptible verities (Figure 6).But in the case of resistant variety, expression levels were significantly higher.
Resistant variety exhibited highest expression level at 2 DPI in resistant variety.At this stage, expressions level of PR 3 gene in resistant variety was 50% more that susceptible one.This suggests greater induction of this gene after pathogen attack (Figure 6).Expression of PR 5 was 21% higher in resistant in resistant variety at 4 DPI as compared to the susceptible one.PR 10 gene represented different trend in this area.Its expression was significantly high in susceptible variety at 0 DPI.But at 4 and 6 DPI, expression levels were higher in resistant variety (Figure 6).

DISCUSSION
Plant mediated interactions between a susceptible host plant and a virulent pathogen is of interest because it provides information about integration of plant defense against many virulent pathogens of same plant.We carried out anatomical and histochemical investigations to understand how sorghum plants with varying susceptibility responded to colletotrichum infection.Colletotrichum causes systemic infection and can induce changes in all aerial parts of the plants.When a pathogen attacks plant, resistance is impaired by localized cell death around infection site (Ma and Shang, 2009).This a rapid reaction initiated by signaling cascade at the attempted infection.In our studies, we observed some cells disintegrated in the cortical region of resistant variety.Necrotic areas were also observed in resistant variety that might limit the further invasion of fungal pathogen.This is attributed to hypersensitive reaction initiated by sorghum plant to limit the further invasion of pathogen.It is therefore reasonable to assume that defense responses make plants to survive even after severe pathogen attack.The ultrastructure features that were observed in microscopic studies confirm this assumption.Systemic invasion of fungal pathogen also induce structural changes in vascular system of host plant.This was observed in the form of tylosis in lumen of meta xylem vessels.
Phenolics, PO, PPO, PAL, CHI and b-1,3-glucanase are directly involved in plant disease resistance (Friend. 1979;Anand et al., 2007;Yao and Tian, 2005;Zhao et al., 2008).Phenolics play an important role in determining resistance or susceptibility of a host to pathogen infection.A resistant variety may contain more phenolics than a susceptible variety (Rubin and Aksenova, 1957;Raghunathan et al., 1958).Present investigation showed that the total phenols were in significant higher amounts in resistant genotypes than that of susceptible one.The similar findings were reported by Arora and Wagle (1985) and Saini et al. (1988).It was also revealed that, both the resistant and susceptible genotypes had an increasing PO activity but in higher amounts in resistant one.The susceptible one showed a decreased PO activity.This is in agreement with the results of Gong et al. (1995).The Anjum et al. 1405 resistant genotypes had a higher PO activity that is suitable to induce systemic resistance in the genotypes, which is characteristic of resistant genotype.This might be why the susceptible genotype had a lower PO activity.PPO and PAL are the major enzyme in phenylpropenoid metabolism leading to the synthesis of lignin, phenols, phytoalexins and other compounds involved in a localized plant resistance process (Shadle et al., 2003).Chitinase and β-1,3-glucanase have been intensively studied and identified as the PR-protein that functions in the plant defense response (van Loon et al., 1998).These enzymes have direct antifungal activity because these decompose β-1,3-glucan and chitin present in the fungal cell wall (Edreva, 2005;van Loon, 1997).β-1,3-Glucanase has also have an indirect effect by degrading the fungal cell wall and then releasing an oligosaccharide elicitor that activates plant defenses.In the present study, when pathogen was applied on sorghum verities, substantial increase in both PPO and PAL activity was noted to a greater extent in resistant variety than that in susceptible one.In the same way, chitinase and β-1,3-glucanase activities were greater in resistant variety.The increase in activities of PO, PPO, PAL, chitinase and β-1,3-glucanase activities in the resistant cultivar correlates with the reduction of disease incidence in resistant variety.Our results are also in agreements with that of Gowda et al. (1989).They also observed an increased PO, PPO and PAL activates in resistant mustard variety against Alternaria blight.In the same way, Gowda et al. (1989) observed increased PPO and PAL activity in resistant sorghum verities against Peronosclerospora sorghi infection process.
During the interaction with pathogens, plants responded with an array of defense mechanisms (Pannecouque and Hoften, 2009).Both structural and chemical barriers are involved which can be constitutive and/or inducible.Comparative histochemical studies of resistant and susceptible plants revealed that deposition of certain defense related structures and biochemical is because of inducible defense response.Lignification of cell wall is considered a potential physical barrier to stop invasion of pathogen in host body.Lignin also restricts feeding of pathogen inside plant body (Ride, 1978).In resistant variety, lignin deposition was highly revealed by both quantification and staining assays.In resistant variety, quantities of lignin were significantly higher than susceptible one.There were fully differentiated xylem vessels having thick lignified secondary walls in resistant plant leaves and stalk.In stalk of resistant variety, lignification of cells in vascular elements spread up to sclerenchyma cells.Moreover, the deposition of guaiacyl lignin was more as compared to syringyl lignin in the cell walls of vascular elements of resistant plant.
All plant parts exposed to the atmosphere are coated with layers suberin that reduce water loss and help block the entry of pathogenic fungi and bacteria (Enstone and Petersone, 1997).Another physical barrier to colletotrichum was deposition of suberin in cell walls of cortex.The brown coloration of cortical cell walls was due to suberin after staining with Sudan III reagent.Despite suberization and lignification of the cell walls, pathogen hyphae penetrated sclarenchymatous cells.This can be attributed to the fact that suberization deposition occurs late after pathogen invasion.
Phenolic acids protect cell wall polysaccharides from enzymatic attacks of pathogen (Hartley et al., 1997).Fungal pathogens produce esterases to break the linkages that cross link cell walls polysaccharides to enhance cell wall degradation (Williamson et al., 1998).Production of phenolics and callose deposition is a frequent response to pathogen invasion in all plant species (Park and Ikeda, 2008;Egea et al., 2001).As was observed in our study, phenolics were deposited in many types of tissues in resistant variety.At some places, cell lumen was completely filled with phenolics globules.Xylem cell walls were intensely deposited with phenolics.Callose deposition was also very much intense in vascular parenchyma of resistant variety.In susceptible variety, phenolic depositions were observed in the whole section under microscope.Quantifications of phenolics also supported results of staining assays.Because in resistant variety, phenolic acids were in significant higher amounts as compared to susceptible one as was evident from calorimetric assay.
Peroxidases are group of isozymes, serving a wide range of functions (Egea et al., 2001).Acidic peroxidases are involved in lignin formation in the cell walls, whereas basic peroxidases are responsible for production of hydrogen peroxide (Collinge et al., 1993).These enzymes are directly related with plant defense response and induced in higher amount when any pathogen tries to invade plant systemically.Peroxidase quantification and staining revealed marked variations in resistant and susceptible sorghum.Resistant plants represented extensive deposition of peroxidases in different tissues of sorghum stalk.Cell walls of dermal tissues and vascular parenchyma were esterified with peroxidases where as in sclerenchyma its deposition was in granular form in resistant variety.
Chitinases play a dual role in the host-pathogen interaction; apoplastic chitinases degrade fungal chitin following initial penetration of the intercellular spaces by the pathogen (Gerhardt et al., 1997).The released chitin then trigger a more generalized defense response resulting in the up-regulation of both apoplastic and vacuolar chitinases, and other defense responses including the hypersensitive response (Gerhardt et al., 1997).We denoted significant higher levels of chitinase in resistant variety.In the same way, rapid increase in levels of chitinases and glucanases was observed by Munch-Garthoff (1997) in wheat under attack of Puccinia graminis f.sp.tritici.
A time-course expression of pathogenesis related genes selected for this study showed that their expression levels were significantly higher in resistant cultivar with slight differences in the case of PR 10 gene.Transcripts of these PR-proteins exhibited a rapid upregulation as early as 2 DPI and reached maximum, then down regulations were noted at later stages.These defense response patterns are identical with those observed by Caldo et al. (2004) for incompatible and compatible interactions involving Mla1-like genes for resistance to powdery mildew on barley; build-up of transcripts of defense-related genes in both susceptible and resistant verities were same except that expression levels decreased during later stages of infection.
Conversely, maximum expression level of PR 2, PR 3 and PR 5 was observed at 2,4 or 6 DPI.Additionally, these genes were expressed at higher levels in resistant variety.Expression patterns of all these genes suggest that these have vital role in sorghum and C. sublineolum pathosystem.Plants produce an array of PR proteins that exhibit differential defense responses against pathogen (Walter, 1992;Niderman et al., 1995).Highest expression level of PR 3 even at 2 DPI suggests that up-regulation of this defense-related protein is among the first defense response affected by pathogen infection in the resistant variety.
Even in susceptible variety, upregulation of expression of PR-protein genes tends to occur at later stages than in resistant reactions and also at lower levels.These delays in up-regulation of defense responses have also been observed in susceptible varieties after pathogen attack (Maleck et al., 2000;Martinez et al., 2003).Some studies have demonstrated that infection of fungal pathogen in resistance and susceptible varieties was similar in initial 7 days after pathogen inoculation.Then, resistance expression resulted in the failure of fungal pathogen to become established in resistant varieties in later days (Ma et al., 2009).Therefore, expression of defenserelated genes would be expected at variable time period after pathogen attack.The very rapid expression of PR-1 and PR-2 genes causes hindrance in the earlier stages of disease cycle.

CONCLUSIONS AND FUTURE RECOMMENDATIONS
Recent study on pathogen mediated induced defense response in sorghum provides us with holistic and physiological view of defense mechanism of a host against a pathogen.As in response to colletotrichum attack, production of lignin, suberin, callose, phenolics and peroxidase was induced in sorghum plant.Based on this information, we should make genetically modified

Figure 1 .
Figure 1.Effect of C. sublineolum on epidermis of susceptible and resistant sorghum verities.(a) Epidermis of susceptible sorghum varieties inoculated with C. sublineolum at 2 DPI.(b) Epidermis of resistant variety was intact at 2 DPI after attack by C. sublineolum.

Figure 2 .
Figure 2. Cytological changes induced by colletotrichum infection in sorghum verities.(a, b) Lignification of epidermal and cortical tisssues in resisitant sorghum variety.(c, d) Destrucyion of tissues in cortical area (bar presents effected area) (d) and formation of hollo space in cortical region (bar presents effected area); EP= epidermis, CO = cortex.

Figure 3 .
Figure 3.Time course studies of changes in defense related biochemicals in resistant and susceptible sorghum verities attacked by C. sublineolum.Results were analyzed by performing ANOVA.(*) significance level at p = 0.5, (**) significance level at p = 0.05.

Figure 4 .
Figure 4. Comparison of localization of phenolics and lignin in cross section of stems of resistant and susceptible sorghum verities.(a, b) Fresh fine sections (40x) were stained with Feric chloride for phenolics and immediately visualized under microscope.(b) Phenolics localization in cortical cells of resistant variety.No phenolics localization was observed in susceptible variety (a).(c, d) Cell walls of vessels elements are esterified with phenolics.(d) Intense phenolics localization in resistant plant's vessel elements.(e, f) Sections stained with Maule reagent (40x) to visualize lignin deposition in vessels elements.(f) Extended deposition of syringyl lignin (SL) round xylem vessels and sclerenchyma in resistant variety as compared to susceptible variety (e).Guacyl lignin (GL) deposition was more only around xylem and phloem vessels in resistant variety (f).phe = Phenolics, p = phloem, mx = meta xylem, px = proto xylem.Sc = sclerenchyma.SL = syringyl lignin.GL = guacyl lignin.

Figure 5 .
Figure 5. Histochemical changes induced by C. sublineolum in stalk of resistant sorghum variety.(a) Peroxidase deposition in cortical tissues visualized at 100x.(b) Radial and tangential cell walls of epidermal cells showing extensive peroxidase deposition (100x).(c) Extensive esterification of peroxidase in cell walls of vascular parenchyma cells (100x).(d) Lignin deposition in cell walls of vascular elements stained with phloroglucinol HCL reagent in infected plant.(e) Suberin deposition in endodermal cell walls visualized by staining with Sudan III.(g) Phenolics globules in lumen of parehchymatous cells.(h) Cell walls of parenchyma esterified with phenolics.(i) Callose deposition in cell walls of vascular elements visualized by analin staining (40x).

Figure 6 .
Figure 6.Time course studies of relative expression of different PR genes in resistant and susceptible sorghum verities attacked by C. sublineolum.Results were analyzed by performing ANOVA.(*) significance level at p = 0.5, (**) significance level at p = 0.05.

Table 1 .
Sequences of primers used for RT-PCR analysis.