Biochemical responses in cotton as diagnostic parameter for resistance against cotton leaf curl virus (CLCuV)

doi:10.21203/rs.3.rs-2237104/v1

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Biochemical responses in cotton as diagnostic parameter for resistance against cotton leaf curl virus (CLCuV)

https://doi.org/10.21203/rs.3.rs-2237104/v1

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Cotton (Gossypium hirsutum L) is one of the most important staple fibrous crops cultivated in India and globally. Its production and quality are greatly hampered by cotton leaf curl disease (CLCuD) caused by cotton leaf curl virus (CLCuV). Therefore, the aim of present study was to investigate biochemical resistance responses in different cotton varieties against CLCuV. Four commercial cotton varieties with susceptible (HS 6 and RCH-134 BG-II) and resistant (HS 1236 and Bunty) response were used to analyse the role of primary (sugar, protein and chlorophyll) and secondary (gossypol, phenol and tannin) biochemical compounds produced by the plants against infection of CLCuV. The resistant cultivars with increased activity of protein, phenol and tannin exhibited as biochemical barriers against CLCuV infection imparting the resistance in cotton cultivars. Whereas, other biochemical compounds including chlorophyll, sugar and gossypol did not show significant role in resistance against CLCuV. Nevertheless, these compounds virtually associated with basic physiological and metabolic mechanisms of cotton plants. Among the primary biochemical compounds, only protein activity proposed as first line of defence in cotton against CLCuV. The secondary level of defence line in resistance exhibited the activity of secondary biochemical compounds phenol and tannins which exhibited significant increase in their level while imparting resistance against CLCuV in cotton.

cotton

cotton leaf curl virus

biochemical

CLCuD

resistance

Cotton (Gossypium hirsutum) is staple fibrous crop cultivated subtropically and seasonally dry areas of northern and southern hemispheres. On the global map India is the largest producer (23%) of cotton (Anonymous, 2019; Barwale, 2016). The global production has shown about 2% decline in production in 2018 due to biotic and abiotic stresses, whereas, overall 6% yield growth of cotton can achieve the global demand of cotton by 2028 (Anonymous, 2019). Cotton leaf curl disease (CLCuD) is the one of most devastating and prominent disease of cotton caused by cotton leaf curl virus (CLCuV) in the Indian subcontinent (Farooq et al., 2011; Monga, 2014; Qadir et al., 2019). The virus belongs to category of begomivirus, a geminivirus (family Geminiviridae) intimately associated with satellite molecules (beta satellite and alpha satellite) and is transmitted by whitefly (Bemisia tabaci Gem.) through circulative persistent transmission in plants (Sharma and Rishi, 2003).

Whitefly may behave different in acquiring the virus, due to biochemical and genetic differences in whitefly races with its host (Yang et al., 2011). Also the compositional differences in host saps could affect the behavior and growth of the insects (Gupta et al., 2010). It is interesting to understand the varieties with different biochemical responses can enhance the understanding of management of CLCuD and its vector whitefly. Various biochemical substances such as primary and secondary compounds including sugar, phenols, phytoalexins, proteins, chlorophyll, phenol, gossypol and tannin are known to influence various physiological processes in plants against diseases and insect pests (Acharya and Singh, 2008; Ajmal et al., 2011; Anuradha, 2014; Beniwal et al., 2006; Bhat, 1997; Borkar and Verma, 1991; Chakrabarty et al., 2002; Govindappa et al., 2008; Hedin and McCarty, 1990; Singh and Agarwal, 2004; Wilson and Smith, 1976). These metabolites have shown association with the plant’s response against biotic stresses, however, little work has been reported for the diseases caused by viruses. We took the biochemical response study in relation to CLCuD in cotton to investigate the response of plants by producing various biochemical primary and secondary metabolites includes sugar, protein, chlorophyll, gossypol, phenol, tannin etc. in cotton which can provide opportunity to study the mechanism involved in the resistance against CLCuV.

1.2.1 Plant Material

Four commercially grown cotton cultivars (two hybrids HS6 & H 1236; two Bt were selected based on the disease incidence and plant types (hybrid and Bt). Out of four, two were resistant (H 1236 and Bunty) against cotton leaf curl virus (CLCuV) and two were susceptible (HS 6 and RCH-134 BG-II). Two Bt hybrid varieties taken were Bunty and RCH-134 BG-II as resistant and susceptible, respectively. Similarly, two non Bt hyrbrid varieties taken were H 1236 and HS 6 have resistant and susceptible response, respectively. Experiment was laid out in plots of 5x4 m² size in randomized block design during the summer (kharif) season. The disease appeared in one month old plants along with the white fly population build up was observed and the disease was allowed to spread under natural epiphytic conditions (Fig. 1a& b). The biochemical compounds viz., sugar (total and reducing sugar), proteins, chlorphyll-a and chlorophyll-b, gossypol, total phenols and tannins analysis was done through standard biochemical methods. Plants were scored on the basis of 1–6 disease grade scale counting 1–2 as resistant (R), 3 as moderately resistant (MR), 4 as moderately susceptible (MS) and 5–6 as susceptible (S) response to CLCuD (Table 1). Representative leaf samples of the CLCuD diseased plants of the four cotton varieties raised in the field conditions. The plants with disease grades 0, 2, 4 and 6 of all four cultivars were picked at 40, 60, 90 and 120 days after sowing to observe the response of plants against disease infection at different plant stages.

Table 1

Disease scale used for grading of CLCuD.
Grade of disease	Reaction response	Plant response
1	R	Thickening of small veins
2	R	Grade 1 + main vein thickening and little leaf curling
3	MR	From top 1/4th of the plant showing leaf curling
4	MS	Upper 1/2 plant affected with leaf curling
5	S	From top 3/4th plant affected with leaf curling
6	S	Severe stunting of plant with leaf curling

1.2.2 Leaf sample preparation

Symptomless leaf samples from resistant cultivars and diseased leaves of different disease grades (2, 4 and 6) were used from susceptible cultivars for the analysis. For fair comparison, leaves without symptom of susceptible cultivars were used as control. Leaves were cleaned with sterilized water to remove any foreign material from the leaf surface and sun dried for 3 days. The dried samples were subjected to oven drying at 60^o C for the complete drying up to 5 days. After drying the leaves were crushed into fine powder for the biochemical analysis, whereas, fresh leaf samples were taken for chlorophyll estimation. The higher disease index of grade-4 in the plants of susceptible cultivars appeared after 30 days of sowing, therefore, biochemical estimation for highly diseased plants was done at second stage of sampling.

1.2.3 Quantitative determination of primary metabolites

1.2.3.1 Sugar determination

To each 100 mg leaf samples (powdered form) taken in the test tubes 5 ml of ethanol (80%) was added. Then test tubes were given the hot water bath for 25–30 minutes at 80^oC and mixed will. As soon as the tubes cooled down to normal temperature and were centrifuged at 4000 rpm for 10 minutes. The supernatant was decanted off into the empty test tubes for each sample making the final volume 10ml by adding distilled water and sugar was estimated (DuBois et al., 1956).

1.2.3.2 Protein determination

To the 100 mg leaves extract taken into 150 ml digestion flask, 10 ml solution of H₂SO₄ and HClO₄ in the ratio of 4:1was poured gently along the walls of flask and was left for 24 hrs undisturbed. After 24 hrs, the flasks were heated on hot plate until solution became colourless and then was allowed to cool at room temperature. By adding distilled water, the final volume was made 100 ml and was transferred to the distillation apparatus. Total protein content was analyzed from the leaves by Kjeldahl method as decribed by AOAC (Helrich, 1990).

1.2.3.3 Chlorophyll-a and chlorophyll-b determination

The determination of chlorophyll-a and b was performed (Hiscox and Israelstam, 1979) and the formulas (Arnon, 1949) used to analyze the total chlorophyll-a and chlorophyll-b were as follows:

Total chlorophyll (mg g^− 1)

=\(\frac{\left(20.2 x {A}_{645}\right)+\left(8.02 x {A}_{663}\right) x V}{\left(1000 x W\right)}\)

Chlorophyll-a (mg g^− 1)

=\(\frac{\left(12.7 x {A}_{663}\right)x \left(2.69 x {A}_{645}\right) x V}{\left(1000 x W\right)}\)

Chlorophyll-b (mg g^− 1)

=\(\frac{\left(22.9 x {A}_{645}\right)+\left(4.69 x {A}_{663}\right)x V}{1000 x W}\)

Where,

A₆₆₃ and A₆₄₅ = absorbance at wavelengths 645 and 663 nm, respectively

V = Volume of solution

W = Weight of sample

1.2.4 Quantitative determination of secondary metabolites

1.2.4.1 Gossypol determination

To the each 500mg powdered cotton leaf samples taken into a 25ml capacity conical flask 10 ml ethyl alcohol (95%) was added. Samples were put under hot water bath for 5 minutes and then filtered into fresh test tubes and centrifuged at 8000 rpm for 15 minutes at 18^oC. The dilution was done by adding 40% ethanol and 1N HCl was added to adjust the pH at 3.0. Using a separating funnel 1.5 ml of diethyl ether at 10^oC was mixed into the content of test tubes. The extract was allowed to evaporate from the tubes until the tubes dried and gossypols was estimated (Bell, 1986).

1.2.4.2 Phenol determination

The method of extract preparation for phenols determination was same as for sugar determination. Standard method (Bray and Thorpe, 1954) was used to estimate phenol content using Folin-Ciocalteu’s agent.

1.2.4.3 Tannin determination

To the 100mg of each leaf extracts taken into oak ridge tubes (10 ml capacity) about 5 ml acetone (70%) was added. Hot water bath at 70^oC for 25–30 min was given to each tube and the content was vortexed to mix all and followed by centrifugation at 4000rpm for 10 minutes after the tubes cooled to the room temperature. The supernatant was decanted off to fresh empty oak ridge tubes and tannin was estimated (Porter et al., 1986).

1.3.1 Sugar (total and reducing)

Maximum total sugar content 14.9 mg g^− 1 was exhibited in the symptomless plants of the susceptible cultivar RCH 134 BG-II at 90 DAS (Fig. 2). Whereas, least sugar 1.5 mg g^− 1 was estimated in highly diseased plants of susceptible hybrid HS 6 at 60 days after sowing (DAS). Decrease in the total sugar was exhibited in resistant cultivars Bunty at 120 DAS.

Similarly, reducing sugar in the healthy plants of the susceptible Bt cultivar RCH 134 BG-II exhibited highest 1.67 mg/g at 90 DAS (Fig. 2). Whereas, lowest 0.07 mg g^− 1 was observed at 60 DAS in the highly diseased plants of the susceptible hybrid HS 6.

1.3.2 Protein

The Maximum protein 26.3 mg g^− 1 was observed at 90 DAS in Bunty, followed by resistant hybrid H 1236 (25.1 mg g^− 1) (Fig. 3). Healthy plants of susceptible cultivars also exhibited increase in the protein content. The susceptible cultivars HS 6 and RCH 134 BG-II with lower disease exhibited more protein as compared to the highly diseased plants. The lowest protein was observed at 120 DAS in susceptible cultivars HS 6 (9.4 mg g^− 1) followed by RCH 134 BG-II (10.5 mg g^− 1).

1.3.3 Chlorophyll (a and b)

Increase in Chlorophyll-a (Chl-a) was observed with the progress in CLCuD symptoms and the crop growth stages. HS 6 and RCH 134 BG-II exhibited the increased Chl-a up to 90 DAS (Fig. 4). Higher Chl-a was exhibited in susceptible cultivars HS 6 (342.8 mg g^− 1) and RCH 134 BG-II (299.1 mg g^− 1) at 90 DAS in the highly diseased plants. Whereas, resistant cultivars H 1236 and Bunty exhibited 223.8 mg g^− 1 and 245.8 mg g^− 1 at 30 DAS, respectively. Chlorophyll-b (Chl-b) remained low in all the cultivars up to30 DAS.

1.3.4 Phenol

Increase in phenol was observed with the plant growth up to 90 DAS, whereas, at 120 DAS phenol content decreased (Fig. 5). Higher phenol content (0.70 mg g^− 1) was observed at 90 DAS in Bunty and H 1236. Whereas, highly diseased HS 6 and RCH 134 BG-II plants exhibited the least phenol (0.25 mg g^− 1) at 90DAS. Highly diseased plants in susceptible cultivars measured the lowest phenol as compared to the resistant cultivars.

1.3.5 Gossypol

Gossypol in all four cultivars was increased up to 90 DAS (Fig. 6). High gossypol 0.75 µg g^− 1 and 0.74 µg/g was measured in susceptible RCH 134 BG-II and the resistant cultivar Bunty, respectively. Gossypol content observed in symptomless susceptible cultivars RCH 134 BG-II and HS 6 was same (0.72 µg g^− 1) at 90 DAS. Whereas, highly diseased plants of the same cultivars exhibited lowest gossypol content.

1.3.6 Tannin

Tannin content increased in all the cultivars up to 60 DAS, thereafter, showed decrease with time at 90 and 120 DAS. High tannin was observed in plants with less disease symptoms as compared to the highly diseased plants (Fig. 7). The healthy leaves of susceptible hybrid HS 6 expressed high tannin (0.76 µg/g) in comparison to highly diseased plants with grade-6 (0.47 µg/g) at 60 DAS. Similarly, symptomless leaves of susceptible Bt cultivar RCH 134 BG-II exhibited 0.93 µg/g tannin in comparison to the highly diseased plants of RCH 134 BG-II had 0.61 µg/g tannin.

The biochemical response in plants during host and virus interaction in the present study proposed the significance of some primary and secondary biochemical compounds involved in the mechanism of resistance against CLCuV. The presence or levels of these metabolites plays significant role to analyse their role in photosynthetic as well as respiratory metabolism of the plants and defense against external entities including herbivores, microbes, viruses (Theis and Lerdau, 2003; Yazaki, 2006). We conducted the present study with the aim to investigate the level of various biochemicals in resistant and susceptible cotton cultivars against CLCuV. Findings of our research revealed difference in levels of sugar, protein, chlorophyll (a & b), phenols and tannins in resistant and susceptible cultivars to get insight of biochemical response during the viral infection in cotton cultivars against CLCuV.

Sugar as part of biochemical activity was studied due to its role as source of energy for various physiochemical activities. The present study exhibits that total sugar in the resistant and susceptible cotton plants did not signify its role in resistance against the virus. Plants exhibited variations in sugar content irrespective of their reaction response to the disease. Some of earlier studies have exhibited high sugar in susceptible reaction (Jayapal and Mahadevan, 1968; Klement and Goodman, 1967; Patil et al., 2010) and in some cases no significance with the disease resistance was observed (Ashfaq et al., 2014). Poor understanding on function of sugar in disease resistance is the limiting factor. Nevertheless, the present study did not find sugar as contributing part in biochemical response against CLCuV, however, it may further be studied as part of host pathogen interaction phenomenon.

The protein biosynthesis in host plant interaction preferably takes place in the incompatible reaction. It had been proposed that high protein content in the infected plant could be due to activation of the host defence mechanisms occurred between host and pathogen (Agrios, 2005). The findings of present study measured high protein content in resistant cultivars H 1236 and Bunty. Comparatively less diseased plants of susceptible cultivars also exhibited increase in protein than the highly diseased plants. The observation on trend of results clearly indicated that protein impart its role in resistance against CLCuV infection. The results of our study are in agreement of previous findings supported the association of protein as one of the defence response against CLCuV in cotton plants (Acharya and Singh, 2008; Beniwal et al., 2006; Siddique et al., 2014).

The stressed plants behave differently for its physiological responses and chlorophyll is one of the contents can be the part of its response in a diseased plant. The present study showed increase in chlorophyll level with the increase in disease index and of plant age. Researchers have elucidated increase in chlorophyll in diseased plant due to its role in intercellular viral movement via symplastic routes within the plant (Zhao et al., 2016). Nevertheless, the findings of our study are not in conformity of the movement of virus, however, the rise in chlorophyll in mature susceptible plants was observed higher than the younger plants in all the cultivars implicating the accumulation of chlorophyll under CLCuV infection. Earlier studies also corroborated rise in chlorophyll in susceptible plants (Ashfaq et al., 2014; Devlin and Witham, 1983; Kandhasamy et al., 2010; Reddy et al., 2005). Limited information was found of work done on role of chlorophyll in disease reaction, however, studies on its mechanism can provide information on involvement of chlorophyll in diseased as well as in mature plants.

In the case of defence response in host-pathogen interactions, phenols perform as most important components having key role in imparting resistance to plant diseases. In our study, the resistant cultivars showed the high phenol content in comparison to susceptible cultivars. Present findings are in close agreement with rise in phenol level in resistant cultivar of cotton against CLCuV (Ajmal et al., 2011) which elucidated the secondary level of defence line in plant. The possible explanation could be that phenolic compounds helps in lignin and suberin synthesis that provides the mechanical strength to host cells as physical barriers against pathogens (Ngadze et al., 2012; Singh et al., 2014) and in the present findings rise in phenolics also infers the resistant reaction against CLCuV. Similar results have been observed in okra (Manju et al., 2021) with high phenol content in resistant reaction against OYVMV. However, the classification of phenolics involved in resistance was beyond the limit of the present study. Though, researchers observed the phenol content could be used as reliable biochemical markers for early selection of genotype resistant to OELCuD (Yadav et al., 2020).

Gossypol is observed as one of the toxic terpenoid aldehyde (TA) compounds against insect pests and a secondary metabolite released in cotton (Heinstein et al., 1979; Widmaier et al., 1980). The CLCuV is transmitted through whitefly, therefore, gossypol level was studied on cotton cultivars in the present experiment. Surprisingly, no indication was observed on change in gossypol content in resistant and susceptible cotton plants against CLCuV. Although, gossypol in cotton is toxic to the insect-pests, nematodes and fungi (Bell, 1986). However, there are opportunities to elucidate the antimicrobial properties and antiviral properties in cotton.

Similarly, tannins are among the most important secondary metabolic compounds that plays important role in plant defense mechanism against diseases and insect pests (Swain, 1979). We also studied the tannin content in the present study and observed that tannin content in susceptible cultivars was decreased with the increase in disease, whereas, resistant cultivars exhibited high tannin content. The corroborated results are in agreement on the role of tannins in cotton cultivars for resistance against CLCuV (Acharya and Singh, 2008; Beniwal et al., 2006).

Through this study we aimed to elucidate the biochemical activity and other changes occurred in the cotton plants infected with CLCuV. The results elucidated clearly that sugar and gossypol are the two natural content in plants that not gave clear information on their level in resistant and susceptible reaction. Whereas, other constituents such as protein, chlorophyll, phenol and tannins indicated that changes in their level can be used as marker of study for resistance against CLCuV. Therefore, the findings of present study offer the understanding of the role of primary and secondary metabolites in resistance against CLCuV in hybrid and Bt cotton.

The present study incited the role of various primary and secondary metabolites such as sugar, protein, chlorophyll, phenol, gossypol and tannin in cotton exhibiting the resistance response against CLCuV. The biochemical response confers the multiple biochemical compounds activity in response to the viral infection in cotton. Resistant cultivars implicated the resistance through activity of proteins, phenols and tannins against CLCuV. The biochemical compounds such as sugar, gossypol and chlorophyll associated with the basic physiological and metabolic mechanisms did not play their role in resistance against CLCuV infection in cotton plant. Among the primary biochemical compounds, activity of protein proposed as first line of defence, whereas, the secondary level of defence line in resistance exhibited the activity of phenol and tannins as most significant in imparting the resistance against CLCuV in cotton. The present study implicated the importance of biochemical study to understand the changes occurred in plants under the biological stress due to a viral infection. Findings of our study revealed the biochemical information to understand the mechanism of action involved in the resistance against CLCuV and can be used as the source of information for the development as well as differentiation of resistance in cotton plants against CLCuV.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgement

The authors express sincere thanks to the Department of Genetics & Plant Breeding, CCS Haryana Agricultural University, Hisar for providing access to biochemical laboratory for the smooth conductance of the study. Authors are also thankful to head of the department of Genetics & Plant Breeding for providing analytical help in the results of experiment. At the end authors extends the acknowledgement to the department of Plant Pathology of the University for providing timely guidance in conducting and writing for the research work.

Ethic approval: It is stated and submitted that the present research article is solely submitted to this journal and is not under consideration with other journal. Publication procedure to this journal has been approved by all the other co-authors.

Conflict of Interest: The authors declare that they have no conflict of interest

Data Availability Statement:

The data that supports findings of this study are available in this article. Colour figures included the result of findings.

Acharya, V.S., Singh, A.P., 2008. Biochemical basis of resistance in cotton to the whitefly, Bemisia tabaci Genn. Journal of Cotton Research and Development. 22(2), 195-199.
Agrios, G.N., 2005. Plant Pathology, 5th ed. Elsevier Academic Press, New York.
Ajmal, S., Perveen, R., Chohan, S., Yasmin, G., Mehmood, M.A., 2011. Role of Secondary Metabolites Biosynthesis in Resistance to Cotton Leaf Curl Virus (CLCuV) Disease. African Journal of Biotechnology. 10(79), 18137-18141.
Anonymous, 2019. Cotton, in: OECD-FAO Agricultural outlook 2019-2028. pp. 217-226.
Anuradha, 2014. Genetical and biochemical basis of cotton leaf curl virus disease in Gossypium hirsutum L. , Department of Plant Pathology. CCS Haryana Agricultural University, Hisar.
Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts: Polyphenoloxidase in Beta vulgaris. Plant Physiology. 24, 1-15.
Ashfaq, M., Khan M.A., T., M., S.T., S., 2014. Role of mineral metabolism and some physiological factors in resistance against urdbean leaf crinkle virus in blackgram genotypes. International Journal of Agricultural Biology. 16, 189‒194.
Barwale, R., 2016. A perspective on cotton crop in India – opportunities and challenges. Cotton Statistics News. 10, 1–3.
Bell, A.A., 1986. Physiology of secondary products Cotton Foundation, Memphis, TN.
Beniwal, J., Sharma, J., Kumar, A., Talwar, G., 2006. Assessment of losses due to leaf curl virus (CLCuV) disease in cotton (Gossypium hirsutum). Journal Cotton Research and Development. 20 (2), 272-279.
Bhat, T., 1997. Source-sink relationship as influenced by late leaf spot in groundnut genotypes. University of Agricultural Science, Dharwad, India.
Borkar, S.G., Verma, J.P., 1991. Dynamics of phenols and diphenoloxidase contents of cotton cultivars during hypersensitive and susceptible reaction induced by Xanthomonas campestris pv. malvacearum. Indian Phytopathology. 44(3), 280-290.
Bray, H.G., Thorpe, W.Y., 1954. Analysis of phenolic compounds of interest in metabolism, in: Anal (Ed.) Glick, D. (Ed.) Meth Biochem. Intersarnae Publishing Inc., New York, pp. 27-52.
Chakrabarty, P.K., Mukewar, P.M., Sheo Raj, Sravan, V.K., 2002. Biochemical factors governing resistance in diploid cotton against grey mildew. Indian Phytopathology. 55(2), 140-146.
Devlin, R.M., Witham, F.H., 1983. Plant Physiology, 4th ed. ed. PWS Publishers, California.
DuBois, M., Gilles, K.A., Hamilton, J.D., 1956. Colorimetric method for determination of sugars and related substances. Annals of. Chern. 28, 350-356.
Farooq, A., Farooq, J., Mahmood, A., Shakeel, A., Rehman, A., Batool, A., Riaz, M., Shahid, M.T.H., Mehboob, S., 2011. An overview of cotton leaf curl virus disease (CLCuD) a serious threat to cotton productivity. . Australian Journal of Crop Science. 5(12), 1823-1831.
Govindappa, N., Hosagoudar, J., Chattannavar, S.N., 2008. Biochemical studies in Bt and non-Bt cotton genotypes against Xanthomonas axonopodis pv. malvacearum. . Journal of Cotton Research Development. 22(2), 215-220.
Gupta, V.K., Sharma, R., Singh, S., Jindal, J., Dilawari, V.K., 2010. Efficiency of Bemicia tabaci (Gennadius) population from different plant-hosts for acquisition and transmission of cotton leaf curl virus. Indian Journal Biotechnology. 9, 271-275.
Hedin, P.A., McCarty, J.C., 1990. Possible roles of cotton bud sugars and terpenoids in oviposition by the boll weevil. J. Chem. Ecol. 16, 757-772.
Heinstein, P., Widmaier, R., Wegner, P., Howe, J., 1979. Biosynthesis of Gossypol, in: Swain T., H.J.B., Van Sumere C.F. (eds) (Ed.) Biochemistry of Plant Phenolics: Recent Advances in Phytochemistry. Springer, Boston, MA, p. 651.
Helrich, K., 1990. Official methods of analysis of the association of official analytical chemists 15th ed. Association of Official Analytical Chemists Inc., Arlington.
Hiscox, J.D., Israelstam, G.F., 1979. A method for the extraction of chlorophyll from leaf tissue without maceration. Canadian Journal of Botany. 57(12), 1332-1334.
Jayapal, R., Mahadevan, A., 1968. Biochemical changes in banana leaves in response of leaf spot pathogens. Indian Phytopathology. 21, 43-48.
Kandhasamy, S., Ambalavanan, S., Palanisamy, M., 2010. Changes in physiology and biochemistry of mottle streak virus infected finger millet plants. Archives of Phytopathology and Plant Protection. 43, 1273–1285.
Klement, Y., Goodman, R.N., 1967. The hypersensitive reaction to infection of bacterial and plant pathogens. Annual Review of Phytopathology. 5, 17-44.
Manju, K.P., Vijaya Lakshmi, K., Sarath Babu, B., Anitha, K., 2021. Morphological and biochemical basis of resistance in okra to whitefly, Bemisia tabaci and okra yellow vein mosaic virus (OYVMV). Journal of Entomology and Zoology Studies. 9(1), 1719-1728.
Monga, D., 2014. Cotton Leaf Curl Virus Disease, in: Technical Bulletin. Publshed by Director, Central Institute for Cotton Research, Nagpur, p. 34.
Ngadze, E., Coutinho, T.A., Icishahayo, D., Van der Waals, J.E., 2012. Role of polyphenol oxidase peroxidise, phenylalanine ammonia lyase, chlorogenic acid and total soluble. Plant Disease. 96, 186-192.
Patil, L.C., Hanchinal, R.R., Lohithaswa, H.C., Nadaf, H.L., Kalappanavar, I.K., Megeri, S.N., 2010. Biochemical relationship in resistant and susceptible cultivars of spot blotch infected tetraploid wheat. Karnataka Journal of Agricultural Sciences. 24(4), 520-522.
Porter, L.J., Hrstich, L.N., Chan, B.G., 1986. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry. 25, 223-230.
Qadir, R., Khan, Z.A., Monga, D., Khan, J.A., 2019. Diversity and recombination analysis of Cotton leaf curl Multan virus: a highly emerging begomovirus in northern India. BMC Genomics. 20, 274.
Reddy, C., Tonapi, V.A., Varanasiappan, S., Navi, S.S., Jayarajan, R., 2005. Influence of plant age on infection and symptomatological studies on Urdbean leaf crinkle virus in urdbean (Vigna mungo). International Journal of Agricultural Sciences. 1, 1–6.
Sharma, P., Rishi, N., 2003. Host range and vector relationship of cotton leaf curl virus from Northern India. Indian Phytopathology. 56, 496-499.
Siddique, Z., Akhtar, K.P., Hameedb, A., Sarwar, N., Imran-Ul-Haqa, Khan, S.A., 2014. Biochemical alterations in leaves of resistant and susceptible cotton genotypes infected systemically by cotton leaf curl Burewala virus. Journal of Plant Interactions. 9(1), 702-711.
Singh, H.P., Kaur, S., Batish, D.R., Kohli, R.K., 2014. Ferulic acid impairs rhizogenesis and root growth, and alters associated biochemical changes in mung bean (Vigna radiata) hypocotyls. Journal Plant Interaction. 9, 267–274.
Singh, R., Agarwal, R.A., 2004. Role of chemical components of resistant and susceptible genotypes of cotton and okra in ovipositional preference of leaf hoppers. , in: Singh, P. (Ed.) Cotton Breeding. Kalyani Publishers, New Delhi, pp. 136-146.
Swain, T., 1979. Phenolics in the environment. Recent Advances in Phytochemistry. 12, 617–640.
Theis, N., Lerdau, M., 2003. The evolution of function in plant secondary metabolites. International Journal Plant Science. 164, 93–102.
Widmaier, R., Howe, J., Heinstein, P., 1980. Prenyltransferase from Gossypium hirsutum. Archives of Biochemistry and Biophysics. 200(2), 609-616.
Wilson, F.D., Smith, J.N., 1976. Some genetic relationship between gland density and gossypol content in G. hirsutum. Crop Science. 16, 830–832.
Yadav, Y., Maurya, P.K., Bhattacharjee, T., 2020. Inheritance pattern of okra enation leaf curl disease among cultivated species and its relationship with biochemical parameters. J. Genet. 99(84), 1-12. https://doi.org/https://doi.org/10.1007/s12041-020-01241-7.
Yang, J.W., Yi, H.S., Kim, H., Lee, B., Lee, S., Ghim, S.Y., Ryu, C.M., 2011. Whitefly infestation of pepper plants elicits defence responses against bacterial pathogens in leaves and roots and changes the below-ground microflora. Journal of Ecology. 99, 46–56.
Yazaki, K., 2006. ABC transporters involved in the transport of plant secondary metabolites. . FEBS Letters. 580, 1183-1191.
Zhao, J., Zhang, X., Hong, Y., Liu, Y., 2016. Chloroplast in Plant-Virus Interaction. Front. Microbiol. 7, 1565.

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Biochemical responses in cotton as diagnostic parameter for resistance against cotton leaf curl virus (CLCuV)

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Abstract

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1.1 Introduction

1.2 Materials And Methods

1.2.1 Plant Material

1.2.2 Leaf sample preparation

1.2.3 Quantitative determination of primary metabolites

1.2.3.1 Sugar determination

1.2.3.2 Protein determination

1.2.3.3 Chlorophyll-a and chlorophyll-b determination

1.2.4 Quantitative determination of secondary metabolites

1.2.4.1 Gossypol determination

1.2.4.2 Phenol determination

1.2.4.3 Tannin determination

1.3 Results

1.3.1 Sugar (total and reducing)

1.3.2 Protein

1.3.3 Chlorophyll (a and b)

1.3.4 Phenol

1.3.5 Gossypol

1.3.6 Tannin

1.4 Discussion

Conclusions

Declarations

References

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