Research Article, J Plant Physiol Pathol Vol: 9 Issue: 2

Biochemical Factors Imparting Resistance to Rust (Puccineapurpurea Cooke) of Sorghum

S.R. Zanjare¹, Y.S. Balgude^2* and Snehal S. Zanjare³

¹Senior Scientist (Pathology), Seed Technology Research Unit, MPKV

²Senior Research Assistant (Pathology), Seed Technology Research Unit, MPKV

³Department of Plant Pathology and Agril. Microbiology, MPKV

*Corresponding Author:

Balgude YS
Senior Research Assistant (Pathology), Seed Technology Research Unit, MPKV, Rahuri Dist, India
Tel: 09890380654
E-mail: [email protected]

Received Date: March 26, 2020; Accepted Date: February 03, 2021; Published Date: February 10, 2021

Citation: Zanjare SR, Balgude YS, Zanjare SS (2021) Biochemical Factors Imparting Resistance to Rust (Puccineapurpurea Cooke) of Sorghum. J Plant Physiol Pathol 9:2.

Copyright: © All articles published in Journal of Plant Physiology & Pathology are the property of SciTechnol, and is protected by copyright laws. Copyright © 2020, SciTechnol, All Rights Reserved.

Abstract

Keywords: Sorghum rust; Rust resistance; Biochemical constituents; Chlorophyll; Peroxidase and polyphenol oxidase

Keywords

Sorghum rust; Rust resistance; Biochemical constituents; Chlorophyll; Peroxidase and polyphenol oxidase

Introduction

Sorghum (Sorghum bicolor L.) is one of the most important grain and fodder crop grown worldwide for food security and believed to be originated from Africa, Nile valley, Central India and has spread through the warmer parts of India, China, South and East Asia and Southern Europe. It ranks fifth after wheat, maize, rice and barley in the list of world’s important cereal crop globally and second after maize in sub-Saharan Africa.

The crop suffers by many more fungal, bacterial and viral diseases viz., leaf blight [Exherohilumturcicum] formerly, Helminthosporiumturcicum, downey mildew [Sclerosporasorghi], crazy top [Sclerosporamacrospora], zonate leaf spot [Gloeocercosporasorghi], grey leaf spot [Cercosporasorghi] mycoplasma. Among these diseases rust of sorghum Pucciniapurpurea Cooke is becoming a serious problem in rabisorghum. It occurs in warmer regions. In India, it occurs in all the states. Severe rust infection also contributes to lodging by reducing leaf area and increasing plant stress [1]. The pathogen form scattered purple, red and flecks on both side of infected leaves and is highly susceptible lines, the flecks may coalesce to form blister like dark reddish brown pustules [2,3].

The rust disease was considered to be minor but now a days it is becoming a major one. Rust is particularly problematic in late-sown crops [4] with yield losses up to 65% resulted from the impact on panicle exertion and grain fill under environmental conditions favourable for early rust development [2]. The disease was noticed in high intensity during rabi 2012-13 in the field of the All India Coordinated Sorghum Improvement Project, MPKV, Rahuri.

In order to minimize losses caused by rust, cultivation of resistant cultivars is one of the cheaper and suitable options over the use of chemicals. Therefore, identification of resistant sources and the factors imparting resistance to rust are needed to be studied thoroughly. Comparative studies on biochemical constituents in R and S genotypes of sorghum during pathogenesis has often helped in understanding the nature and mechanism of used as basis for identification of R genotypes. Now, a little information is available regarding factors imparting rust resistance and their activities. The present study attempts to identify the biochemical factors which help in identification of traits responsible for resistance to rust of sorghum.

Materials and Methods

Estimation of biochemical components such as chlorophyll (chlorophyll ‘a’, chlorophyll ‘b’ and total chlorophyll) content, sugar content (reducing sugar, non-reducing sugar and total sugar) total phenols, peroxidase activities and polyphenol activities was carried out in five rust resistant (RSV2390, RSV2394, RSV2395, RSV2393 and RSV2383) and five rust susceptible (RSV2388, RSV2381, RSV2400, P. Anuradha and M 35-1) genotypes of sorghum. An experiment was carried out during 2018-19 at the Department of Plant Pathology and Agriculture Microbiology, Mahatma PhuleKrishi Vidyapeeth, Rahuri under the controlled glass house condition. Seed of the each genotype were sown in the plastic pots. All the plants were inoculated at the 4 to 5 leaf stage with an inoculum concentration of urediniospores (Pucciniapupurea) and incubated at 20-25°C under high RH (>90%) for 24 hours after 30 days of sowing (DAS) [5]. Simultaneously, similar sets of all the five genotypes were sown in pots separately under rust free environment in another glasshouse for comparison. Rust severity was recorded at 60 days after sowing (DAS) using ‘0 to 9 scale’ suggested by [6]. Further per cent disease index (PDI) was calculated using the formula given by [7].

Sampling for biochemical studies was done at 30 days after inoculation (DAI) from both the sets. Standard procedures were followed for estimation of different biochemical constituents from the leaf portion i.e. chlorophyll content [8] soluble protein by the method of [9] reducing sugars by Nelson Somogyi‟s method [10] total sugars by [11] total phenols by using Folin-Denis reagent as described by [12] peroxidase activity and polyphenol oxidase activity [13]. Non reducing sugar was calculated by subtracting reducing sugars from total sugars.

Results and Discussions

Severity of sorghum in different soybean genotypes: Rust severity assessed at 60 DAS on all the genotypes revealed that it differed significantly as far as genotypes, crop growth stages (days) and their interaction are concerned. The genotypes RSV2390, RSV2394, RSV2395, RSV2393 and RSV2383showed highly resistant reaction to rust. However, maximum rust severity was recorded in RSV2388, RSV2381, RSV2400, P. Anuradha and M 35-1 at 60 DAS (Table 1).

Table 1: Screening of sorghum genotypes in glasshouse to rust disease.

Biochemical studies

Infection by pathogen brings about lot of changes in respiratory pathway and photosynthesis which are the vital processes taking place in the plant leading to wider fluctuation in biochemical components. This in turn alters the resistance of the host. Some studies on biochemical components in resistant and susceptible sorghum genotypes were carried out as described in material and methods and the results are presented here under. Biochemical analysis in resistant and susceptible sorghum genotypes was carried out 60 days after sowing (DAS) i.e. 30 days after inoculation (DAI) to understand their role in resistance or susceptibility of rust pathogens.

Chlorophyll

The results on chlorophyll ‘a’, chlorophyll ‘b’ and total chlorophyll content as influenced by rust analyzed at 30 DAI are presented in Table 2 and Figures 1-3. In general, levels of Chlorophyll content were higher at 30 DAI in healthy plants but lower under inoculated condition. Per cent decrease in all the three chlorophyll components over healthy leaf and R genotypes was observed in both R and S genotypes after inoculation.

Table 2: Chlorophyll ‘a’ ‘b’ and total chlorophyll content in different resistance and susceptible sorghum genotypes as influenced by rust disease rust disease.

The data on chlorophyll ‘a’ indicated that it was high in healthy plants, but decreased under inoculated condition. The chlorophyll ‘a’ differed significantly among the resistant and susceptible genotypes. The genotype RSV2395 recorded highest chlorophyll ‘a’ in healthy stage (1.693 mg/g fresh wt.) and also in inoculated stage (1.587 mg/g fresh wt.) followed by genotype RSV2381 in healthy condition. However, the lowest chlorophyll content was recorded in the genotype RSV2393 under inoculated condition (0.659 mg/g fresh wt.). The mean chlorophyll ‘a’ was more in the resistant genotypes, at both healthy and inoculated condition than in susceptible genotypes. It was also noted that there was decrease in the per cent mean chlorophyll ‘a’ in inoculated condition over healthy in both resistant genotype (9.78%) and susceptible genotypes (13.04%).

In case of chlorophyll ‘b’ and total chlorophyll, same genotype RSV2395 recorded highest chlorophyll “b” (0.511 mg/g fresh wt.) and also total chlorophyll (2.204 mg/g fresh wt.) in healthy stage and decreases in inoculated condition. However, the lowest chlorophyll „b‟ was recorded in the genotype P. Anuradha under inoculated (0.112 mg/g fresh wt.) and lowest total chlorophyll content was found in genotype RSV2393 (0.775 mg/m fresh wt. The mean chlorophyll ‘b’ and total chlorophyll was more in the resistant genotypes, at both healthy and inoculated condition and decreases in susceptible genotypes. It was also noted that there was decrease in the per cent mean chlorophyll ‘b’ at inoculated condition over healthy in both resistant and susceptible genotypes i.e. 46.95% and 47.68%, respectively and in case of total chlorophyll it was 17.81% and 19.73%, respectively. Total chlorophyll content is presented in Table 2 and Figure 1.

The study revealed that chlorophyll a, chlorophyll b and total chlorophyll contents were decreased due to the foliar infection by Puccineapurpurea in sorghum. In case of chlorophyll ‘a’, mean chlorophyll content was 1.167 mg/g fresh wt. in healthy and 1.048 mg/g fresh wt. in inoculated condition. In case of chlorophyll ‘b’ mean chlorophyll content in healthy was 0.354 mg/g fresh wt. and 0.242 mg/g fresh wt. in inoculated and 1.521 mg/g fresh wt. in healthy and 1.281 mg/g fresh wt. in inoculated in case of total chlorophyll.

The phenomenon of reduction of chlorophyll has been reported by many workers attributing to various reasons. Amongst them, [14] reported decrease in chlorophyll content due to infection in several host pathogen systems and Heath [16] reported a change in the ultra-structure of chloroplast in rusted cowpea leaves [15] studied the chlorophyll content and mineral composition of downy mildew affected chlorotic leaves of sorghum and found reduction in content of chlorophyll ‘a’ and chlorophyll ‘b’ content [17] studied the effect of late leaf-spot disease on chlorophyll content in different groundnut varieties and reported substantial loss of chlorophyll in susceptible varieties than that of partially resistant varieties. [18] reported that the total chlorophyll content was higher in healthy leaves than inoculated leaves with Phaeoisariopsispersonata and also observed that the chlorophyll content was higher in resistant cultivar and low in susceptible groundnut cultivar. [19] observed that the chlorophyll content was more in healthy leaves than the Phomopsis infected leaves of tea plants. They also observed that the chlorophyll content was slight more in tolerant than susceptible cultivar. [20] reported that the chlorophyll content decreased due to the infection of Alternariahelianthi. The rate of decrease was more in susceptible genotypes than resistant genotypes.

Sugars Content

The results in respect of reducing sugars, non reducing sugars and total sugars as influenced by rust disease recorded are given in Table 3 and Figures 4-6. The results revealed that significant difference existed among the genotypes. There was decrease in reducing sugars, non reducing sugars and total sugars observed under infected condition in all the resistant and susceptible sorghum genotypes than in healthy.

Table 3: Reducing, non reducing and total sugar content in resistance and susceptible sorghum genotypes as influenced by rustdisease.

Genotype RSV2390 recorded the highest reducing sugars in both at healthy condition (14.93 mg/g fresh weight) and inoculated stage (12.960 mg/g fresh wt.) followed by RSV2393 in healthy and in inoculated stage. However, the lowest was recorded in the genotype RSV2400 at healthy (6.399 mg/g fresh wt.) and in infected condition (5.077 mg/g fresh wt.). The mean reducing sugars was more in the resistant genotypes, at both healthy and inoculated condition when compared with mean reducing sugars of susceptible genotypes. Also it was noted that there was decrease in the per cent mean reducing sugar at inoculated condition over healthy in both resistant and susceptible genotypes (16.97% and 18.16% respectively).

Decrease in the non reducing sugars content was observed under infected condition in all the resistant and susceptible sorghum genotypes. During investigation it was found that, RSV2393 recorded the highest non reducing sugars in both at healthy condition (23.515 mg/g fresh weight) and inoculated stage (21.236 mg/g fresh wt.) followed by genotype RSV2395 in both healthy and in inoculated stage. However, the lowest was recorded in the genotype P. Anuradha at healthy (10.740 mg/g fresh wt.) and in infected condition (9.242 mg/g freshwt.). The mean non reducing sugars was more in the resistant genotypes, at both healthy and inoculated condition when compared with per cent mean non reducing sugars of susceptible genotypes. It was also noted that there was decrease in the per cent mean of non reducing sugar content at inoculated condition over healthy in both resistant and susceptible genotypes (10.31% and 18.48%, respectively).

Decrease in the total sugars content was observed under infected condition in all the resistant and susceptible sorghum genotypes.

Genotype RSV2393 recorded the highest total sugars in both at healthy condition (35.683 mg/g fresh weight) and inoculated stage (31.429 mg/g fresh wt.) followed by genotype RSV2395 in both healthy and in inoculated stage. However, the lowest was recorded in the genotype P. Anuradha at healthy (19.037 mg/g fresh wt.) and in infected condition (16.048 mg/g freshwt.). The mean total sugars was more in the resistant genotypes, at both healthy and inoculated condition when compared with per cent mean total reducing sugars of susceptible genotypes. It was also noted that there was decrease in the per cent mean of total reducing sugar content at inoculated condition over healthy in both resistant and susceptible genotypes (12.69% and 18.36%, respectively).

Sugars acts as precursor for synthesis of phenolics, phyto alexins, lignin and cellulose which play an important role in defense mechanism of plants against invading pathogens. Generally, high levels of total sugars, reducing sugars and non-reducing sugars in the host plants are stated to be responsible for disease resistance. Difference in sugar level between resistant and susceptible genotypes was due to inherent character of the genotypes. It was observed that there was decrease in the reducing sugar content in the resistant and susceptible genotypes which was ranging from 3.06 to 30.55 per cent in case of non reducing sugar, 6.66 to 28.64 percent in case of total sugar and 9.07 to 23.58 per cent.

These results are in conformity with [21] reported that in rust resistant genotype of sorghum, the quantity of reducing sugar was more at all stages of crop growth than the moderately resistant and susceptible genotype. [22] observed the reduction of total and reducing sugar in Pucciniagraminis f.sp. tritici affected stem and leaf sample of wheat. [23] reported that downy mildew susceptible sorghum varieties contained less of total and non reducing sugars than the multiple resistant cultivars viz., SB 2413 and SB 2415. [24] stated that the multiple foliar disease resistant sorghum genotypes possessed higher content of sugar as compared to susceptible ones.

Soluble Proteins Content

The observations on soluble protein as influenced by rust disease recorded at different stages are presented in Table 4 and Figure 7 It was evident that significant difference existed among the genotypes. Decrease in soluble protein was observed under infected condition in all the resistant and susceptible sorghum genotypes. Genotype RSV2383 recorded the highest soluble protein in both at healthy condition (49.79 mg/g fresh weight) and inoculated condition (42.90 mg/g fresh wt.) followed by genotype RSV2390 in healthy and in inoculated stage. However, the lowest was recorded in the genotype M35-1 at healthy (27.44 mg/g fresh wt.) followed by genotype RSV 2388 in infected condition (22.21 mg/g fresh wt.).

Table 4: Protein content, total phenol content, Peroxidase activity and Polyphenol oxidase activity in different resistance and susceptible sorghum genotypes as influenced by rust disease.

The mean soluble protein was more in the resistant genotypes, at both healthy and inoculated condition when compared with mean soluble protein of susceptible genotypes. Also it was noted that there was decrease in the per cent mean soluble protein at inoculated condition over healthy in both resistant and susceptible genotypes (17.78% and 21.95%, respectively).Mean soluble protein content was more in resistant genotypes than the susceptible genotypes. In general, it was noticed that decrease in the soluble protein content in response to foliar infection crop growth ranging from 25.23 to 14.98 per cent. The rate of decrease in the soluble protein content in response to rust disease infection was more in susceptible genotypes.

Results on protein content in different resistance and susceptible sorghum genotypes as influenced by rust disease are in agreement with [25] reported changes in protein content in sorghum leaves infected by Helminthosporiumturcicum Pass. The protein content in healthy and infected leaves was 0.31 and 0.39 per cent, respectively in ten days old plant and 0.24 and 0.02 per cent, respectively in 60 days old plants.[24] reported that the multiple foliar disease resistant sorghum genotype possessed higher protein content compared to those of susceptible genotype. [26] recorded more protein content in resistance and moderately resistant varieties of groundnut than susceptible one. [27] reported that the protein content was more in healthy leaves than infected leaves of cotton genotypes as influenced by the Xanthomonasaxonopodispv. malvacearum. [28] observed that the healthy leaves of both resistant and susceptible genotypes showed more protein content than grey mildew infected leaves of each genotype in cotton.

Total Phenol

Results of the study on total phenols as influenced by rust disease recorded in Table 4 and Figure 8 Decrease in the total phenols was observed under infected condition in all the resistant and susceptible sorghum genotypes. Genotype RSV2383 recorded the highest total phenols in healthy condition (0.911 mg/g fresh weight) and in at inoculated stage (0.842 mg/g fresh wt.) followed by RSV2394 in healthy and in inoculated stage. However, the lowest was recorded in the genotype RSV2400 at healthy (0.521 mg/g fresh wt.) and P. Anuradha in infected condition (0.413 mg/g fresh wt.).

The mean total phenols was more in the resistant genotypes, at both healthy and inoculated condition while compared with per cent mean total phenols of susceptible genotypes. It was noted that, there was decrease in the total phenols at inoculated condition over healthy in both resistant and susceptible (17.93% and 28.15%, respectively). High concentration causes an instant lethal action by a general tanning effect while, low concentration causes gradual effect on the cellular constituent of the parasite. If the concentration does not occur at toxic level, the inhibition will be obviously slow.

There is significant positive correlation between phenolic content and disease resistance. In this study, lower levels of phenols were observed in diseased plant at both the stages of all susceptible genotypes. It was also observed that decrease in phenol content ranged from 8.141 to 29.47 per cent. Mean phenol content in healthy genotypes was 0.610 mg/g fresh wt. where as in inoculated genotypes it was 0.499 mg/g fresh wt. The rate of decrease in the total phenolcon tentinresponsetotherustdiseaseinfectionwasmoreinsusceptibleascom paredto healthy ones.

Similar results were obtained by [29] found higher level of phenolics in resistant sorghum genotypes to Macrophominaphaseolina (Maubl) Ashby than in susceptible ones. [30] reported that healthy hybrids (CSH 6 and 148) resistant to Helminthosporiumturcicum Pass. contained comparatively large amounts of total phenols than in the susceptible cultivars Swarna and Neerujola. [24] reported that multiple foliar disease resistant sorghum genotypes recorded higher content of phenols as compared to susceptibleones.

Peroxidase Activity

The observations on peroxidase activity as influenced by rust disease recorded in Table 4 and Figure 9. The results revealed that significant difference existed among the genotypes. There was an increase in the peroxidase activity was observed under infected condition in all the resistant and susceptible sorghum genotypes. The genotype RSV2390 recorded the highest peroxidase activity in healthy condition (4.12 units/mg/g fresh wt.) and increases in inoculated stage (4.61 units/mg/g fresh wt.) followed by RSV2383 in both healthy and in inoculated stage. However, the lowest was recorded in the genotype RSV2400 at healthy (2.82 units/mg/g fresh wt.) and genotype M35-1 in infected condition (3.28 units/mg/g fresh wt.)

It was noted that there was increase in the per cent mean peroxidase activity at inoculated condition in both resistant and susceptible genotypes (19.34% and 11.83% respectively) Peroxidase oxidizes phenolics to highly toxic quinines and hence, has been assigned a role in disease resistance. The increased activity of peroxidase was observed in resistant and susceptible genotypes which was ranged from 7.89 to 29.62 per cent. Mean peroxidase content in healthy genotype was 3.395 units/g fresh wt. whereas 4.045 units/g fresh wt. in inoculated condition.

These results were in agreement with [31] who reported peroxidase and polyphenol oxidase activity in downy mildew resistant (DMRS 1 and DL 3) and susceptible (DMS 652) sorghum cultivars in ten days old seedlings inoculated with Peronosclerosporasorghi(Weston) Shaw. The activity of both the enzymes were analysed at 15, 30 and 60 hr after inoculation. They found that peroxidase activity was very low in healthy leaves of all the three sorghum lines. Inoculation with P. sorghi increased the peroxidase activity to varying degrees in all the three sorghum lines with the highest increase in DL-3. Also, [32] observed higher activities of peroxidase and polyphenol oxidase in the resistant cultivar (No 179) of sunflower than the susceptible cultivar (EC 68414) following infection with P. helianthi. [27]observed that the healthy leaves of resistant and susceptible cotton plant exhibited less polyphenol oxidase activity and peroxidase activity as compared to infected leaves of cotton as influenced by grey mildew disease.

Polyphenol Oxidase Activity

The data on polyphenol oxidase activity as influenced by rust disease recorded at different stages are presented in Table 4 and Figure 10. There was increase in the polyphenol oxidase activity which observed under infected condition in all the resistant and susceptible sorghum genotypes. The genotype RSV2383 recorded the highest polyphenol oxidase activity in inoculated condition (0.091 units/g fresh wt.) followed by RSV2390 in inoculated stage (0.090 units/mg/g fresh wt.) followed by RSV2395 in inoculated condition. However, the lowest wasrecorded in the genotype in both at P. Anuradha healthy (0.044 units/g fresh wt.) followed by genotype M 35-1 (0.050 units/g fresh wt.) in healthy condition.

The mean polyphenol oxidase activity was more in susceptible genotype as compared to resistant genotypes and mean polyphenol oxidase activity in healthy genotypes were 3.359 units/g fresh wt. and 4.045 units/g fresh wt. in inoculated condition. Also it was noted that there was increase in the per cent mean polyphenol oxidase activity at inoculated condition in both resistant and susceptible genotypes (18.18% and 19.50%, respectively).

Results of the present study, on polyphenol oxidase activity in different resistance andsusceptiblesorghumgenotypesasinflue ncedbyrustdiseaseareinagreementwith [33]. They observed that the polyphenol oxidase activity was relatively more in tolerant cultivars than susceptible cultivars in groundnut as influenced by leaf spot pathogen. [32] observed higher activities of peroxidase and polyphenol oxidase in the resistant cultivar (No 179) of sunflower than the susceptible cultivar (EC 68414) following infection with P. helianthi. In infected leaves of the susceptible cultivar there was an increase in peroxidase activity but the ratio of peroxidase activity decreased during the later period of infection. [27] observed that the healthy leaves of resistant and susceptible cotton plant exhibited less polyphenol oxidase activity and peroxidase activity as compared to infected leaves of cotton as influenced by grey mildew disease.

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