Volume 2, Issue 2 (2015) Tropical Plant Research

7/23/2019 Volume 2, Issue 2 (2015) Tropical Plant Research

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www.tropicalplantresearch.com 72Received: 27 December 2014 Published online: 30 June 2015

ISSN (E): 23491183ISSN (P): 23499265

2(2): 7277, 2015

Research article

I n vitroantioxidant activity of selected Ganodermaspecies found

in Odisha, India

Ashutosh Rajoriya, Sushri Shant Tripathy and Nibha Gupta*

Plant pathology and Microbiology division, Regional Plant Resource Centre, Bhubaneswar, Odisha, India

*Corresponding Author: [email protected] [Accepted: 25 April 2015]

Abstract: Four species of Ganoderma were collected from different forests of Odisha and

analysed for the presence of antioxidant components. Data revealed the presence of DPPH free

radical scavenging properties ranged between 91.6495.51 % in these species. Ganoderma

applanatum exhibited a studied for the same. Both the Ganoderma species exhibited higher

amount of tannin content too. Alkaloid content was ranged in 3.665.51 mg.gm-1 in these four

species. Present study exhibited the presence of good higher amount of total phenol, ascorbic acid,

carotenoid whereas lycopene and ergosterol content was found to be maximum in Ganoderma

lucidium as compared to other mushroom species amount of flavonoid content in Ganoderma

tsugae followed by G. lucidium and G. applanatum. This preliminary exploratory data of the

present study indicated that Ganoderma species are good source of antioxidant components.

Further extraction and purification of this component may throw light on its role as an

antioxidant in Ganodermasp.

Keywords:Mushrooms - Ganoderma- Antioxidants - Flavonoids - Ergosterol.

[Cite as: Rajoriya A, Tripathy SS & Gupta N (2015)In vitroantioxidant activity of selected Ganodermaspecies

found in Odisha, India. Tropical Plant Research 2(2): 7277]

INTRODUCTION

Mushrooms are well known for their therapeutic properties like antibacterial, antifungal, antiviral,

antitumour, immunomodulating, antiallergic, antiatherogenic, hypoglycemic, anti-inflammatory and

hepatoprotective activities (Ferreira et al. 2010, Lindequist et al. 2005). These bioactivities are mainly due to -

glucans, phenolics, vitamins, organic acids and trace elements (Cheung et al. 2003, Khatuna et al. 2013,

Iwalokun et al.2007).

Mushrooms are also considered as home remedy to protect from various diseases elicited by oxidative stress

or free radical stress (Chen et al.2012). It is mainly due to the antioxidants which are widely distributed in

mushrooms (Jose & Janardhanan 2000, Liu et al. 1997, Barros et al. 2007). The family Ganodermataceae

presents polypore kind of Basidiomycetous fungi having a double walled basidiospore (Donk 1964).

Basidiocarps of this genus possess a shiny surface which is associated with the pilocystidia embedded in an

extracellular melanin matrix (Moncalvo 2000). Ganoderma species are ubiquitous in the world with varied

characteristics, such as different shapes, size and color (red, black, blue/green, white, yellow, and purple) of the

fruit body, specific host and geographical origin (Zhao & Zhang 1994, Woo et al. 1999, Upton 2000).

Ganoderma lucidum (Curtis) P.Karst. (Common names: Reishei, Lingzhi) is a species of Basidiomycetes

that belongs to Ganodermataceae of Polyporales (Yang et al. 2000). Smina et al.(2011) reported various types

of antioxidants from the Ganodermawhich can reduce oxidative damage by directly scavenging free radicals

generated in the cell. Ganoderma lucidium shows bioactivity against hypertension, bronchitis, arthritis,

neurasthenia, hepatopathy, chronic hepatitis, nephritis, gastric ulcer, tumorogenic diseases,

hypercholesterolemia, immunological disorders, scleroderma, cardiovascular disease, AIDS and cancer (Sliva

2003), beside G. lucidium, G. tsugaeand G. applanatumis also used for the bioactivity which in turn can be

well utilized for the health benefits, many reports are available regarding their anticancer, antioxidant,


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www.tropicalplantresearch.com 73

antibacterial, antiviral behavior (Mau et al.2005a, Mau et al.2005b, Jeong et al. 2008, Kim et al. 1998, Rym et

al.1999).

The main objective of the present work is to explore the antioxidant properties of some Ganodermaspecies

found in Odisha.

MATERIALS AND METHODSCollection and identification

Healthy, fresh and succulent macrofungi were collected from tropical moist deciduous and semi evergreen

forests from different forest divisions of Odisha Macroscopic and microscopic examination of different parts

such as pileus, stipe, veil, ring, volva, lamellae and gills etc. were focused to identify species. All of the assays

were carried out using the entire mushroom. Mushrooms were cleaned and subsequently dried in the oven at

50C for about 4 hrs. All of the dried mushrooms were ground to fine powder (100 mesh) and stored in air tight

plastic container at room temperature till all the analysis.

Antioxidant components

Total Phenolic Content: Total phenolic content in the wild mushrooms were estimated through folin phenol

method as described by Singleton and Rossi (Moradali et al. 2006). The optical density was measured at 765

nm using spectrophotometer (Analytic Jena).Ascorbic acid content:The ascorbic acid content in the wild mushrooms was determined by volumetric method

(Singleton & Rossi 1965). The amount of ascorbic acid in mg 100 g-1

sample is calculated by using formula;

0.5 mg/V1 ml V2 /5100/ weight of the sample100, when V1 is the standard ascorbic acid consumed

against dye.

Flavonoids:The flavonoid content in dried sample was estimated by using aluminum chloride colorimetric

technique and expressed in terms of quercetin equivalents per gram (Harris & Ray 1935).

Beta carotene and Lycopene:The concentration of -carotene and lycopene in mushroom extracts was estimated

by spectrophotometer following Nagata and Yamashita (1992), Chang et al.(2002) & Barros et al.(2007).

Carotenoid:The carotenoid content was estimated in 500 mg of dried mushroom powder treated with 10 ml of

80% acetone and centrifuged at 3000 rpm for 10 minutes at 4C. The quantity of carotenoid was calculated

(Arnon 1949).Tannins: Tannic acid was served as a standard and tannin content was estimated at 760nm according to

Schanderl (1970) and expressed in mg.gm-1.

Alkaloids: Total alkaloid was estimated after extraction with glacial acetic acid and ethanol and precipitated

with Draggendroffs reagent. The residue treated with sodium sulfide and thiourea solution and optical

density was measured at 435 nm for alkaloid estimation (Srividya & Mehrotra 2003).

Ergosterol:Total ergosterol was estimated with chloroform and methanol mixture followed by NaCl2 and glacial

acetic acid treatment. Finally sterol content was estimated by using ferric chloride reagent and measuring

absorbance at 550 nm (Sadasivam & Manickam 1996).

Antioxidant assay

Free radical scavenging activity: The DPPH free radical scavenging activity was estimated in the methanolic

extracts by colorimetric method (Chan et al. 2007). 1 ml of methanolic extract was added with 2 ml ofDPPH solution (1:2) and incubated for 30 min. in dark after vigorous shaking. Absorbance was measured at

517 nm and scavenging activity of each extract was calculated.

Reducing power ability: Each mushroom extract (0.54 mg.ml-1) in methanol (2.5 ml) was mixed with 2.5 ml of

200 mM sodium phosphate buffer (pH: 6.6) and 2.5 ml of 1% potassium ferricyanide, and the mixture was

incubated at 50C for 20 minutes. After 2.5 ml of 10% trichloroacetic acid was added, the mixture was

centrifuged at 2000 rpm for 10 minutes. The upper layer (5 ml) was mixed with 5 ml of deionized water and

1 ml of 0.1% ferric chloride, and the absorbance was taken at 700 nm (Analytik Jena) spectrophotometer.

Ec-50 value was calculated in mg.ml-1at 0.5 optical density against reagent blank (Oyaizu 1986).

Ferric Antioxidant Reducing Power (FRAP):100 l of the methanolic extract was mixed with 3 ml of FRAP

reagent and incubated in the room temperature in dark for 10 minutes and finally absorbance was read at 593

nm (Analytik Jena) spectrophotometer. FRAP value was expressed in terms of mg AEAC.gm-1

of sample(Benzie & Strain 1996, Athavale et al. 2012).


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RESULTS AND DISCUSSIONS

In the present study four species of Ganoderma (i.e. G. lucidium, G. tsugae, G. applanatumand Ganoderma

sp.) were collected and analyzed for the antioxidant properties. Radical scavenging activity was seen best in the

G. tsugae(95.51%) and Ganoderma sp. (94.43%) at the IC50 value of 12 mg.ml -1and 10 mg.ml-1respectively

(Table 1). Thus the assay used to test the radical scavenging activity shows that scavenging activity is directly

proportional to the concentration of the sample. Since free radicals are the causal agents for the oxidative stress

and different aliments Ganodermasamples used in the present studies can with stand for the health benefits.

Presence of ergosterol in range of 0.0280.45 mg.gm-1 in these Ganoderma species is also revealed in the

present studies (Mattila et al.2002).

Table 1. Representing antioxidant activity of different wild Ganodermaspecies from Odisha, India.

Species DPPH (%) IC50 (mg.ml-1

) FRAP (mg AEAC.gm-1

)

Ganoderma lucidium 93.19 9 2.290.14

Ganoderma tsugae 95.51 10 1.820.01

Ganoderma applanatum 91.64 6 2.430.05

Ganoderma sp. 94.43 12 0.240.04

The phenolic content of Ganoderma sp. was ranged in 911.60 mg.gm-1 whereas highest flavonoid content

was observed in G. tsugae (0.840.37 mg.gm-1). A very high amount of Ascorbic acid i.e. 1.400.01 and

1.10.16 mg.gm-1was found in G. applanatum and G. lucidium, respectively (Table 2). More or less similar

quantities of carotenoid content ranged in 3.484.65 mg.gm-1

were observed in all Ganoderma sp. studied

except G. applanatum (7.430.29 mg.gm-1

). All the four species of Ganoderma showed a good amount of

alkaloid and tannin content.

Table 2. Analysis of antioxidant components of wild Ganodermaspecies from Odisha, India.

S. No. Parameters G. lucidium G. tsugae G. applanatum Ganoderma sp.

1 Total Phenolic 9.000.30 9.000.10 11.600.20 11.400.10

2 Flavonoids 0.630.15 0.840.37 0.620.13 0.380.06

3 Ascorbic acid 1.100.016 0.700.017 1.400.011 0.600.0174 Carotenoids 4.47 0.05 4.650.73 7.430.29 3.480.49

5 -Carotene 3.630.19 1.660.16 3.300.77 1.600.43

6 Lycopene 0.2240.029 0.1880.002 0.1770.014 0.0430.004

7 Ergosterol 0.490.067 0.470.067 0.440.094 0.280.044

8 Alkaloids 3.660.10 4.501.15 4.400.25 5.510.16

9 Tannins 2.290.14 1.820.01 2.430.05 0.240.04

The presence of phenolic compounds in Ganodermaconfirms the observations of Rawat et al.(2013) and

Celik et al.(2014). Phenolic compounds are responsible factors of antioxidant properties in many mushrooms

and plants (Ferreira et al.2009, Barros et al.2008). Data revealed good radical scavenging properties by these

mushroom species may be due to good phenolic content also (Table 2). Flavonoids are the group of phenolic

compounds which were assumed to be accumulated only in the plants but not by the animal or any fungi

(Ferreira et al.2009) however a good amount of flavonoid content was recorded in present studies. Flavonoid

content in all the four species of Ganodermaranged from 0.84 to 0.38 mg.gm-1

which is also supported by the

studies of Logananthan et al.(2010) and Barros et al. (2008) and can be compared with the edible mushroom

varieties.Due to the presence of flavonoid content these species may be considered as curing agent for various

cardiovascular, anti-proliferative, detoxification and anti-inflammatory type of diseases (Le 2002).

Findings of carotenoid content in the Ganodermaspecies in good amount made them important at par with

fruits and vegetables (Mangels et al. 1993). A higher amount of Beta carotene and lycopene was exhibited in

these species and corroborated with the reports of Robaszkiewicz et al.(2010), Pal et al.(2010) and Celik et al.

(2014). Ascorbic acid is considered to exert a protective role against many oxidative stress related ailments such

as cardiovascular, cancer, neurodegenerative problems and cataract (Halliwell 1996). Ascorbic acids ranged

from the 1.400.60 mg.gm-1of the sample which is comparable to the reports of Barros et al.(2008). Tannins

are polyphenolic compounds responsible for various bioactivities viz. antimicrobial, antioxidative and antitumor


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activities (Hatano et al.2006, Yoshizawa et al.1987, Okuda & Ito 2011). Data presented in table 2 regarding

tannin content in the four species of Ganodermaranged between 2.290.24 mg.gm-1is well compared with the

results of Puttaraju et al.(2006) and Onuoha et al. (2010).

The present study suggests the Ganoderma species are the good source of antioxidant compounds. Further,

elucidation and purification of these compounds may lead towards the findings of new class of antioxidants

useful for the development of health care agent.

ACKNOWLEDGEMENTS

The financial assistance obtained from Ministry of Environment and Forests, Govt. of India (Project no. 22-

24/2010 CS.I) is gratefully acknowledged.

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www.tropicalplantresearch.com 78Received: 12 February 2015 Published online: 30 June 2015

ISSN (E): 23491183ISSN (P): 23499265

2(2): 7881, 2015

Research article

Additions to lichen flora of Jammu and Kashmir, India

Reema Goni and Namrata Sharma*

Department of Botany, Jammu University, Jammu & Kashmir, India

*Corresponding Author:[email protected] [Accepted: 10 June 2015]

Abstract: The paper deals with the addition of 44 species to the knowledge of lichen flora of

Jammu & Kashmir state, India. The corticolous (25) lichens exhibit their dominance in the area of

study followed by saxicolous (16), lignicolous (2) and terricolous (1) forms.

Keywords:Diversity - Hot spot - Doda - Himalayas - Altitude.

[Cite as: Goni R & Sharma N (2015) Additions to lichen flora of Jammu and Kashmir, India. Tropical Plant

Research 2(2): 7881]

INTRODUCTION

State of Jammu and Kashmir lies between 32 17' and 36 58' north latitudes and 73 26' and 80 30' east

longitudes and exhibits varied climate associated with wide altitudinal deviations. As such it provides a wide

range of substrates for growth and colonisation of lichens. It is often called as Hot Spot for lichen diversity in

India (Sheikh et al. 2006a). Inspite of this, lichen collection started late here in early thirties of last century

(Smith 1931). Comprehensive accounts came later. First of this series was by Sheikh et al. (2006a, b). The

authors listed a total of 279 lichen species from the state. The collections made by these authors were from

diverse areas, which included Achabal, Pahalgam, Kukernag, Verinag, Baltal, Neh Nar Glacier, Mamal village;

Kangan, Shankaracharya Hill, Ganderbal, Zaberwan, Sonmarg, Harwan Garden, Prang Garden, Shalimar

Garden, Gulmarg, Baba Rishi, Yarikah Pine Forest, Tangmarg, Drang Forest, Khilanmarg, Ferozpur;

Awantipura, Pingalgam & Goosu, Yusmarg, Pulwama and Budgam all from Kashmir region; Nandini Hill,

Jammu University Campus, Mansar Lake, Nagrota, Vijaypur, Nud, Banihal, Kishtwar, Chinta valley, Ramtund

Forest area, Kaplash & Bhaderwah from Jammu region and Hemis National Park from Ladakh region. 30

species from Mansar-Surinsar wildlife Sanctuary of Jammu were added to this list later in 2009. Solan (2010)

enumerated 18 species belonging to 15 genera and 10 families from Ramnagar Wildlife Sanctuary, Jammu.

Later inclusions were 77 species from Kishtwar, Rajouri and Jammu districts by Sheikh et al. (2013), 18 species

from Nandini Wildlife Sanctuary by Goni et al.(2013), 24 species from Zanskar valley, Ladakh by Kumar et al.

(2014) and 25 species from Kargil district of Ladakh region by Rahim et al. (2014). A checklist of lichen flora

of Jammu and Kashmir by Goni et al. (2015) revealed the occurrence of 356 species of lichens belonging to 35

families and 91 genera.

These wide collections however do not include several areas of the state. One such area is district Doda of

Jammu region lying between 3308' to 3324' North latitude and 7523' to 7549' East longitude and coveringan area of 4,500 km

2with an altitude varying from 914 to 4267 meter above mean sea level. This wide district

characterized by a wide range of climatic and physiographic conditions, providing thereby different type of

habitats. Our survey of this area led us to a collection of about 500 lichen samples. Complete identification of

these resulted in the addition of 44 species to the lichen flora of J&K state. This communication depicts these

additions.

MATERIALS AND METHODS

During the period 20112014, various localities in the Doda district were surveyed for the collection of

lichens. The specimens were collected from base to chest height of tree trunk and from all other available

substrates.

Identification of the collected specimens was done using relevant keys and literature (Awasthi 1991, 2000,2007, Nayaka, 2004, Divakar & Upreti, 2005). In the laboratory, the specimens were investigated


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Goni & Sharma (2015) 2(2): 7881.


morphologically, anatomically and chemically. Chemistry was studied with the help of colour spot tests and thin

layer chromatography. The colour tests were performed with the usual chemical reagents which included 5%

KOH solution (K-test), aqueous solution of calcium hypochlorite (C-test) and paraphenylene diammine (PD

test). Lichen substances were investigated with the thin layer chromatography (TLC) in solvent system A (180

toluene: 60 dioxane: 8 acetic acid) using the technique of Culberson (1972) and Walker and James (1980).


The paper deals with a survey of district Doda of J&K state and enumerationof 44 species belonging to 27

genera and 18 families which are new additions to the lichen flora of J&K state (Table 1). Among the various

growth forms collected presently crustose forms (29) were the dominant followed by fruticose (7), foliose (6)

and squamulose (2) forms. On the basis of substratum type, 25 species have been observed to be corticolous,

while 16 species have been recorded as saxicolous forms. Lignicolous and terricolous forms are represented by

2 and 1 species respectively. Graphidaceae(7 species) was the dominant family followed by Parmeliaceae and

Ramalinaceae(6 species each), Teloschistaceae(4 species); Lecanoraceae and Physciaceae (3 species each);

Cladoniaceae (2 species); Verrucariaceae and Lecideaceae (2 species); Acarosporaceae, Arthoniaceae,

Coniocybaceae, Lichinaceae, Megasporaceae. Pannariaceae, Peltigeraceae, Pyrenulaceae and Trapeliaceae (1

species each). Caloplaca appressa, Cladonia didyma, Dimelaena oreina, Lichinella

cribellifera, Melaneliatominii, Porpidia albocoerulescens, Verrucaria laevata, Verrucaria margacea and Xanthoparmelia

antleriformisare some of the major lichen species collected from the study area (Fig. 1).

Table 1. Lichens of Doda district (J&K State) enumerated with their growth forms and substratum from Doda district of J&K state.

S. No. Taxa Growth form Substrate Place of collection Altitude

1 Acaropspora tominiana Magnusson Crustose Saxicolous Nalthi 1795 m2. Arthonia radiata (Pers.) Ach. Crustose Corticolous Sartingal 1700 m3. Aspicilia dwaliensis Rsnen Crustose Saxicolous Khanpura 1594 m4. Bacidia alutacea (Kremp.) Zahlbr. Crustose Corticolous Dandi 1603 m

5. Bacidia medialis (Tuck. ex Nyl.) Zahlbr. Crustose Corticolous Dandi 1607 m6. Bacidia submedialis (Nyl.) Zahlbr. Crustose Corticolous Dandi 1606 m7. Buellia disjecta Zahlbr. in Hand. Mazz. Crustose Saxicolous Drudoo 1329 m

8. Buellia palnienis S.R. Singh & D.D. Awasthi Crustose Saxicolous Batogra 1905 m9. Caloplaca appressa Wetmore & Krnefelt Crustose Saxicolous Phigsoo 1350 m10. Caloplaca granularis (Mll. Arg.) Zahlbr. Crustose Corticolous Khani 2124 m11. Caloplaca lithophila H. Magn. Crustose Saxicolous Gath 1198 m12. Caloplaca ochroplacaPoelt & Hinter. Crustose Saxicolous Chilli 1785 m

13. Cladonia didyma (Fe) Vain. Fruticose Lignicolous Traun 1975 m14. Cladonia fruticulosaKremp. Fruticose Lignicolous Traun 1990 m

15 Chaenotheca chrysocephala (Ach.) Th. Fr. Crustose Corticolous Traun 1994 m16. Dimelaena oreina (Ach.) Norman Sqaumulose Saxicolous Bhella 1142 m17. Graphis arecae Vain. Crustose Corticolous Tanta 2100 m18. Graphis dendrogrammaNyl. in Cromb. Crustose Corticolous Dandi 1675 m19. Graphis epimelaena Mll. Arg. Crustose Corticolous Khanpura 1594 m20. Graphis leptocarpa Fee Crustose Corticolous Dandi 1687 m

21. Graphis lineola Ach. Crustose Corticolous Udrana 1580 m22. Graphis longiramea Mll. Arg. Crustose Corticolous Dandi 1626 m23. Graphis scripta (L.) Ach. Crustose Corticolous Tanta 2089 m24. Fuscopannaria subgemmascansUpreti & Divakar Squamulose Corticolous Puneja 1900 m25. Lichinellacribellifera (Nyl.) P. P. Moreno & Egea Fruticose Saxicolous Shiwa 908 m26. Lecanora pseudargentataLumbsch Crustose Corticolous Dareja 1780 m27. Lecanora sulphurescens Fe Crustose Saxicolous Dareja 1780 m28. Lecidella elaeochroma (Ach.) M. Choisy Crustose Corticolous Bhalla 1678 m29. Melanelia tominii(Oksner) Essl. Foliose Corticolous Tanta 1980 m

30. Mycobilimbia hunana(Zahlbr.) D.D. Awasthi Crustose Saxicolous Seri 1271 m31. Parmelia squarrosa Hale Foliose Corticolous Sartingal 1851 m

32. Parmelienella wallichiana(Taylor )Hale Foliose Corticolous Drudoo 1324 m33. Parmotrema pseudotinctorum(Abbayes) Hale Foliose Corticolous Seri 1391 m

34. Peltigera collina (Ach.) Schrad. Foliose Terricolous Amira nagar 1723 m35. Porpidia albocoerulescens(Wulfen) Hertel & Knoph Crustose Saxicolous Khanpura 1595 m


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36. Pyrenula mamillana (Ach.) Trevis Crustose Corticolous Khani 220 m37. Ramalina farinacea (L.) Ach. Fruticose Corticolous Traun 2013 m38. Ramalina intermedia (Delise ex Nyl.) Nyl. Fruticose Corticolous Puneja 1900 m39. Ramalina subfarinacea (Nyl.) Nyl. Fruticose Corticolous Pranoo 1945 m40. Trapelia placodioidesCoppins & P. James Crustose Saxicolous Sartingal 1987 m

41. Usnea orentalisMotyka Fruticose Corticolous Traun 2034 m42. Verrucaria laevata Ach Crustose Saxicolous Kursari 1857 m43. Verrucaria margacea(Wahlenb.) Wahlenb. Crustose Saxicolous Kothi 1657 m44. Xanthoparmelia antleriformis(Elix) Elix & J. Johnst. Foliose Saxicolous Shiwa 870 m

Figure 1. Some major lichens: A, Caloplaca appressa; B,Cladonia didyma; C,Dimelaena oreina; D,Lichinella cribellifera:E, Melanelia tominii; F, Porpidia albocoerulescens; G, Verrucaria laevata; H, Verrucaria margacea; I, Xanthoparmelia

antleriformis.

ACKNOWLEDGEMENTS

The authors are grateful to the Head, Department of Botany, University of Jammu; Dr. C.S. Nautiyal,

Director and Dr. D.K. Upreti, Head, Lichen Lab, CSIR-National Botanical Research Institute, Lucknow forproviding necessary laboratory and library facilities. We also thank UGC for financial support for field trips

from SAP grant to the Department of Botany.

REFERENCES

Awasthi DD (1991) A key to Microlichens of India, Nepal and Srilanka.Biblioth. The Lichenologist40: 1-337.

Awasthi DD (2000)Lichenology in Indian Sub-continent.Bishen Singh Mahendra Pal Singh, Dehradun, India.

Awasthi DD (2007)A Compendium of the Macrolichensfrom India, Nepal and Sri Lanka. Bishen Singh

Mahendra Pal Singh, Dehradun, India.

Culberson CF (1972) Improved conditions and new data for the identification of lichen products by a

standardized thin- layer Chromatographic method.Journal ofChromatography72: 113-125.


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Goni & Sharma (2015) 2(2): 7881.


Divakar PK & Upreti DK (2005)Parmeloid Lichens in India(A Revisionary study).Bishen Singh Mahendra Pal

Singh, Dehradun, India.

Goni R, Raina AKP & Magotra R (2013) Lichen diversity in Nandini Wildlife Sanctuary, Jammu (J&K).

Phytotaxonomy13: 106108.

Goni R, Raina AKP, Magotra R & Sharma N (2015) Lichen flora of Jammu and Kashmir State, India: An

updated checklist. Tropical Plant Research 2(1): 6471.

Kumar J, Rai H, Khare R, Upreti DK, Dhar P, Tayade AB, Chaurasia OP & Srivastava RB (2014) Elevational

controls of lichen communities in Zanskar valley, Ladakh, a Trans Himalayan cold desert. Tropical Plant

Research 1(2): 4854.

Nayaka S (2004) Revisionary studies on Lichen genus Lecanora sensu lato in India, Ph. D. Thesis. Avadh

University Faizabad, Uttar Pradesh, India.

Rahim A, Raina AK & Hussan A (2014) Lichen diversity of Kargil town and its adjoining areas, J&K.

International Journal of Current Research5(14): 14.

Sheikh MA, Upreti DK & Raina AK (2006a) An enumeration of Lichens from three Districts of Jammu and

Kashmir, India.Journal of Applied Biosciences32(2): 189-191.

Sheikh MA, Upreti DK & Raina AK (2006b) Lichen Diversity in Jammu and Kashmir, India. Geophytology

36(1&2): 69-85.Sheikh MA, Raina AK & Upreti DK (2009) Lichen Flora of Surinsar-Mansar Wildlife Sanctuary, Jammu and

Kashmir.Journal of Applied and Natural Sciences1(1): 79-81.

Sheikh MA, Raina AK & Hussan A (2013) A preliminary observation of lichen flora in three districts of Jammu

& Kashmir.International Journal of Current Research 5(4): 96668.

Smith A L (1931).Lichens from Northern India. Trans.British Mycological Society16: 128-132.

Solan S, Mehta, KA & Magotra R (2010) A catalogue of lichens of Ramnagar Wildlife Sanctuary, Jammu

(J&K).Phytotaxonomy10: 134138.

Walker FJ & James PW (1980) A revised guide to the microchemical techniques for the identification of lichen

products.Bulletin of British Lichenology Society46: 13-29.


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www.tropicalplantresearch.com 82Received: 25 February 2015 Published online: 30 June 2015

ISSN (E): 23491183ISSN (P): 23499265

2(2): 8284, 2015

Research article

Two species of Zygnemopsis (Skuja) Transeau from West

Bengal, India

Nilu Halder

Department of Botany, Raja Peary Mohan College, Uttarpara-712258, Hooghly, W.B., India*Corresponding Author:

[email protected] [Accepted: 12 June 2015]

Abstract: In the present paper, two species of the genus Zygnemopsis viz. Zygnemopsis

pseudolahaulensis andZygnemopsis benghalensis of Zygnemaceae under the order Zygnematales

of Chlorophyta had been morpho-taxonomically described first time from Hooghly district in West

Bengal, India. These algal species had been collected from ponds of this district. The above

mentioned two taxa were new reports from the district and also the second report from state ofWest Bengal, India.

Keywords:New report - Chlorophyta -Zygnemopsis- Hooghly district - West Bengal.

[Cite as: Halder N (2015) Two species of Zygnemopsis(Skuja) Transeau from West Bengal, India. Tropical

Plant Research 2(2): 8284]

INTRODUCTION

The members of Zygnemaceae are filamentous and prefer to grow in winter season and, form climax in early

summer season. Finally they form zygospores or aplanospores during reproductive phase in summer as free

floating condition. Zygnemopsis (Skuja) Transeau, contains about 55 species all over the world and all the

known species are isogamous (Yin-xin 1994). Previously, few works had also been described the members of

Zygnemaceae from the state and country. Martens (1869) first recorded the occurrence of Zygnemaceae fromRaniganj area of West Bengal. In the year 1959, a commendable taxonomic work had been carried out by

Randhawa on the members of Zygnemaceae and he published his findings in the monograph 'Zygnemaceae'

from India. Some other noteworthy publications on Zygnemopsis included: Das (1962), Patel & Kumar (1971,

1977), Prasad & Kumari Vijay (1977), Sharma & Kargupta (1986) and Chalotra et al. (2013). As since 1986 no

morpho-taxonomic report on Zygnemopsis was found from West Bengal, keeping view this paucity of

taxonomic information the present study was undertaken from this area. The aim of the present study was the

exploration of biodiversity of Zygnemaceae and documentation of green filamentous algal species to prepare

algal data bases of this state in future. Human anthropogenic activities, loss of algal habitats and increase of

pollution level in water bodies might be responsible for rare occurrence of this alga in this state. Therefore, a

proper sustainable management is required for functioning aquatic ecosystems and maintains biodiversity of

phyco-flora.


Algal samples were collected in plastic and glass containers from two places viz.ponds at Jirat (N 23-12' E

88-45') and Somrabazar (N 23-13' E 88-43') of Hooghly district, West Bengal. Detail study was made by

examining specimens under Olympus microscope (Model-CH20i) for determination of species. Samples were

preserved in 4% formalin. Identification of different taxa was accomplished with the help of authentic literatures

viz.Randhawa (1959), Patel & Kumar (1971, 1977) and Sharma & Kargupta (1986). Each currently accepted

name has been provided with its author(s) name. Water temperature (C) was recorded using Zeals (U.K.)

Mercury thermometer on the spot. pH of water was measured with the help of portable digital pH meter (Merck,

Germany, Model No. 320). NO3-N, PO43-, dissolve oxygen (DO), biological oxygen demand (BOD), chemical

oxygen demand (COD), SO42-

, total soluble salts (TSS), total dissolve solids (TDS) and total alkalinity were

measured by using UV-VIS spectrophotometer (CECIL CE- 7200) according to the method of APHA (2005).

All parameters in ecological notes were expressed in mg l-1

except pH and temperature (C).


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Halder (2015) 2(2): 8284.



A total number of two algal species of the genus Zygnemopsis (Skuja) Transeau, 1934 viz. Zygnemopsis

pseudolahaulensis Sarma & Kargupta andZygnemopsis benghalensis Sarma & Kargupta of Zygnemaceae under

the order Zygnematales of Chlorophyta had been morpho-taxonomically described first time from Hooghly

district in West Bengal, India.

Morpho-taxonomic description

Order: Zygnematales

Family: Zygnemaceae

1.Zygnemopsis pseudolahaulensis Sarma & Kargupta in Hydrobiologia 139:249, figs. 1-16, 1986 (Fig. 1A)

Free floating, greenish-brown, vegetative cells 15.2 to 17.2 m broad and 54.2 m to 76.2 m long;

chloroplasts two, nearly rounded; pyrenoid single in each cell; zygospores not formed; aplanospores get swollen

and filled with pectic-cellulose materials; ovoid to sub-globose and almost filling the sporangium laterally; 20.0

to 25.0 m broad and 29.0 to 33.0 m long and, brown; outer spore wall smooth, thin and median spore wall

wrinkled or with wavy corrugations.

Habitat: Pond water at Jirat.

Collection No: 1001; Dated: 29.02.2011

Ecological Notes: Jirat, water temperature: 21C; pH: 7.6; NO3-N: 0.20; PO43-: 0.32; DO: 6.4; BOD: 4.8; COD:

110.0; SO42- : 6.0; TSS: 110.0; TDS: 162.0; Total alkalinity: 128.0

Occurrence:Rare

Significance: Primary producer in aquatic bodies.

Figure 1. A,Zygnemopsis pseudolahaulensis Sarma & Kargupta; B,Zygnemopsis benghalensis Sarma & Kargupta.

2.Zygnemopsis benghalensis Sarma & Kargupta in Hydrobiologia 139: 247, figs. 1-11, 1986 (Fig. 1B)

Free floating, greenish-brown, vegetative cells 11.5 to 21.5 m broad and 40.5 m to 96.5 m long;

chloroplasts two and stellate; pyrenoid single in each cell; zygospores not formed; aplanospores get swollen and

filled with pectic-cellulose materials; ovoid to cylindric ovoid and almost filling the sporangium laterally; 16.0

to 31.2 m broad and 33.2 to 35.0 m long and, brown; outer spore wall smooth and median spore wall

irregularly slightly wavy.

Habitat: Pond water at Somrabazar.

Collection No: 1003; Dated: 29.02.2011

Ecological Notes: Somrabazar, water temperature: 21C; pH: 7.4; NO3-N: 0.15; PO43-: 0.28; DO: 6.6; BOD:

4.8; COD: 120.0; SO42-

: 6.4; TSS: 98.0; TDS: 156.0; Total alkalinity: 132.0


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Halder (2015) 2(2): 8284.


Occurrence:Rare

Significance: Primary producer in aquatic bodies.

Reporting new species from any area as new record or recollecting the species have its own importance in

floristic works (Singh et al.2014, Srivastava et al.2014). Das (1962) described a new species ofZygnemopsisas

Z. queensefrom Calcutta. Patel & Kumar (1971) made observation on morphological and cytologicalcharacteristics of Zygnemopsis godwardensesp. nov. from Gujarat and followed by earlier work they (1977)

also recorded three new species of Zygnemopsisviz.Z. chohanensis,Z. dharampurenseandZ. tricarinata while

studying of Zygnemaceae from Gujarat, India. Prasad & Kumari Vijay (1977) identified a new species of

ZygnemopsisasZ. vermaiifrom India. Sharma & Kargupta (1986) first described three species of Zygnemopsis

viz.Zygnemopsis benghalensissp. nov.,Zygnemopsis pseudolahaulensissp. nov. andZygnemopsis scorbiculata

sp. nov. from West Bengal, India. Chalotra et al. (2013) made morpho-taxonomic studies on this genus

occurring in fresh water bodies in Jammu and Kashmir and reported three species namely Z. splendens, Z.

tiffaniana andZ. minutawhich were new to algal taxonomy of Jammu. Apart from the above mentioned studies,

it was the second report since its original description by Sarma & Kargupta (1986) from Birbhum district, West

Bengal, India. This study might be helpful to explore diversity and occurrence of these species in aquatic

ecosystems.

The morpho-taxonomic study of two species of Zygnemopsis viz.Zygnemopsis pseudolahaulensis and

Zygnemopsis benghalensis under the order Zygnematales of Chlorophyta will be provided valuable taxonomic

information in respect of systematic position, author citation, description, habitat, collection number along with

dates, ecological note, significance and occurrence for the first time from this area.

ACKNOWLEDGEMENTS

The author expressed his deepest sense of gratitude and sincere thanks to Dr. S.N. Sinha, Dept. of Botany,

University of Kalyani, Nadia, West Bengal for providing opportunity to work under his guidance.The author is

also grateful to Dr. R.K. Gupta, BSI, Howrah for his kind co-operation.

REFERENCES

APHA (2005) Standard methods for the examination of water and waste water (21st ed.). American Public

Health Association, Washington, DC, New York.

Chalotra P, Gaind M & Anand VK (2013) Morpho-taxonomic studies on the genus Zygnemopsis (Skuja)

Transeau, 1934 (Chlorophyta) occurring in fresh water bodies of Jammu and Kashmir.International Journal

of Innovative Research & Development 2(4): 266272.

Das CR (1962) A new species ofZygnemopsis (Skuja) Transeau, 1934 from Calcutta 1961. National agricultural

fair. Current Science 31: 255.

Islam AKMN (1972) New and rare species of some green algae from Bangladesh. Nova Hedwigia23(4): 655

663.

Martens GV (1869) Beitrgezur Algen-Flora Indiens.Flora 52: 23338.

Patel RJ & Kumar CKA (1971) Morphological and cytological studies in Zygnemopsis godwardense sp. nov.

Phycological society of India10(12): 1217.

Patel RJ & Kumar, CKA (1977) Zygnemataceae of Gujarat, India. III. Zygnemopsis (Skuja) Transeau.Actabotanica Indica5(1): 2024.

Prasad BN & Kumari Vijay CMRS (1977)Zygnemopsis vermaii a new species from India. Geophytology7(1):

5860.

Randhawa MS (1959) Zygnemataceae. Indian Council of Agricultural Research, New Delhi, pp. 1478.

Singh A, Tiwari V & Mohan J (2014) Chroococcales in river Gabga at JajmauGhat, Kanpur. Tropical Plant

Research1(1): 2830.

Srivastava N, Suseela MR & Toppo K (2014) Fresh water cyanobacteria of Sai River near Lucknow, Uttar

Pradesh. Tropical Plant Research1(2): 1116.

Yin-xin W (1994) A new species of Zygnemopsis(Zygnemataceae) and its reproduction cycles.Chinese Journal

of Oceanology and Limnology12(2): 174180.


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www.tropicalplantresearch.com 85Received: 10 March 2015 Published online: 30 June 2015

ISSN (E): 23491183ISSN (P): 23499265

2(2): 8589, 2015

Research article

The effect of sodium silicate and silica nanoparticles on seed

germination and growth in the Vicia fabaL.

Ghaffar Roohizadeh*, Ahmad Majd and Sedigheh Arbabian

Department of Biology, Faculty of Biological Sciences, Islamic Azad University, Tehran, Iran

*Corresponding Author:

[email protected] [Accepted: 17 June 2015]

Abstract: Silicon is the second most common element in soil that has beneficial effects on living

and non-living increase stress tolerance in plants. It can lead to increased production and product

quality, reduce evaporation of perspiration, increased stimulation of some antioxidant enzymes and

decreased sensitivity to some of the fungus. The effects of silicon on seed germination and growth

of the bean (Vicia fabaL.) were investigated. The seeds of plant were treated by 0 (as control), 1.5and 3 mM of sodium silicate and silica nanoparticles. There were three repeats for all treatments.

The test results showed that seeds treated with sodium silicate concentration of 3 mM significant

difference in the percentage of germination than the control no significant difference in the rate of

germination of seeds treated compared to control was observed. Hypocotyl length and the

flowering of all treated plants were significantly different compared to control. The highest

flowering in plants treated silica nanoparticles was observed at a concentration of 1.5 mM. Only

plants treated silica nanoparticles with a concentration of 3 mM significant difference in diameter

than the control plants. According to the test results can be deduced the effect of silicon

nanoparticles in the form of sodium silicate and silica increases the percentage of germination and

growth of broad bean.

Keywords:Nano silica - Sodium silicate - Growth - Seed germination - Vicia faba.

[Cite as: Roohizadeh G, Majd A & Arbabian S (2015) The effect of sodium silicate and silica nanoparticles on

seed germination and some of growth indices in the Vicia fabaL. Tropical Plant Research 2(2): 8589]

INTRODUCTION

Vicia fabaL. is an annual plant of family Fabaceaewith 80110 cm height. The flowers of broad bean are

white with black or purple spots. The seeds are sheathed and the fruits, seeds and flowers have medical usages.

Vicia fabaL.is hetero fertilized with 2n=12. Because of possessing of high percentage of proteins (3034%) it

is an important crop. After oxygen, silica is the second structural element in the earth which is non-mobile in the

plants. Although silica is not necessary for plants, higher plants need it to have optimum growth (Richmond &

Sussman 2003, Ma et al. 2004, Currie & Perry 2007). The most effect of silica on plants, is related to the

resistance against biotic and abiotic stress (Ma & Yamaji 2006, Liang et al. 2007). As the cell wall of plants

prevents the entrance of elements into cells, the Nano particles which have less diameter than the pores of cell

wall, therefore can easily cross the pores. Nano particles in the leavess surface enter the plants through the

stomata and or base of hairs and is then transported to the different organs (Nair et al. 2010). Silica plays

important role in the tolerance against salt stress (Zhu et al. 2003), manganese toxicity (Shi et al. 2005), boron

toxicity (Gunes et al. 2007) and cadmium toxicity (Vaculik et al. 2009, Shi et al. 2010) via changing the activity

of antioxidant enzymes. This study the effect of sodium silicate and silica nanoparticles on seed germination,

hypocotyle length, stem diameter, and amount flowering of the broad bean is done.


In order to assess ment the effects of silica nano particles and sodium silicate, on seed germination of broad

bean (Vicia fabaL.), the samples were grown in greenhouse. Before cultivation, the impact seeds were sterilized

in 5% hypochlorite sodium solution. The seed then were washed up by deionised water. In each pot 2 seedswere cultivated. Solution containing 0 (as control), 1.5 and 3 mM of nano particle of silica and sodium silicate,


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Roohizadeh et al. (2015) 2(2): 8589.


were used in the experiment. The temperature of greenhouse was adjusted to 222 C (at night) and 252 C (at

day). The relative humidity was 44%. The samples were treated for 65 days. SPSS Ver.16 was used for

comparing of the means using Duncan test at P


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Roohizadeh et al. (2015) 2(2): 8589.


with different concentrations of silicon 1.5 mM silica nanoparticles with a concentration of 3 mM, no significant

differences were observed (Fig. 2A).

Stem diameter

Study results showed that Stem diameter silica nanoparticles stabilization period, hearts treated plants only 3

mM significant difference in Stem diameter than the control. The plants treated with concentrations of 1.5 and 3mM sodium silicate significant difference from plants treated silica nanoparticles with a Stem diameter of 1.5

and 3 mM showed Highest and lowest Stem diameter of the treated plants at a concentration of 3 mM sodium

silicate and silica nanoparticles with a concentration of 3 mM compared to control plants (Fig. 2 B).

The amount of Bolting

The results are significant differences in the rate of flowering in plants treated with concentrations of 1.5 and

3 mM sodium silicate and silica nanoparticles as compared to the control the amount flowering in all treatments

was higher than control. The increasing concentration of 1.5 mM or nanosilica flowering of treatment (Fig. 3).

Figure 3.The effect of sodium silicate and silica nanoparticles the amount of Bolting. (Means SE and P < 0.05)

DISCUSSIONS

Germination and seedling establishment of the most critical stages in the life cycle of the plant is The most

important stages of germination, including water absorption, enzymatic activity, the growth of the embryo, seed

coat and tears seedling emergence is the results of the experiments, the researchers suggest that chemical

treatments can stimulate seed germination in many species of plants. Zhu et al. (2010) reported calcium,

gibberellic acid, ascorbic acid, ethanol to speed up germination and Mohammadi et al. (2009) described the

effect of salysyk acid and gibberellic acid on seed germination rate of lentil. The positive effects silicon

attributes tomato plant germination (Haghighi et al. 2012) and soybean (Li et al. 2004) have also been reported.

Mozaffarian et al. (2011) & Manzer et al. (2013) demonstrated that silica nanoparticles improve seed

germination in tomato plants. Increased percentage of soybean germination by combining nanoparticles of

silicon and titanium has also been observed (Lu et al. 2002) this study, the addition of silicon to form sodium

silicate and silica nanoparticles become improves seed germination.

Effects of sodium silicate and silica nanoparticles on growth indices

The present study, reported that the use of silicon improves the growth of root, stem and leaves plant. This

effect may be due to the prominent role of silicon in improving plant water status (Romero-Aranda et al. 2006).

The benefits of using silicon indirect effects such as increased capacity and efficiency of photosynthesis,

transpiration and thus reduce shoot growth related (Liang 2003). Samuels et al. (1993) showed that in the

presence of silicon increases plant growth by improving the mechanical strength of stems and leaves on light

absorption and photosynthetic capacity of the plant is increased. Kamindiou et al. (2010) observed the effects of

silicon on morphological characteristics and growth of gerbera flowers in the greenhouse cultivation, and the

positive effects of the use of silicon as the medium Na2Sio3 the plant height, the thickness of the shoot the size

flowers flowering reported. Reezi et al. (2009) found that adding 50 mg-lpotassium silicate or nutrient Hot Lady


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Roohizadeh et al. (2015) 2(2): 8589.


Rose cut increases the number of flowers. The results of our study showed that the use of sodium silicate and

silica nanoparticles increases the length of the hypocotyl, stem diameter and the amount of Bolting of the Vicia

faba.

CONCLUSION

The results of this study, sodium silicate and silica nanoparticles can affect the germination of seeds. Thus,in cases such as seeds that grow with the problem of different concentrations of these substances can be used to

facilitate and accelerate the germination of seeds and increase the efficiency of their applications. It can also be

used these to help plant growth and ultimately better performance.

ACKNOWLEDGEMENTS

The authors are thankful of all laboratories personnel Mahmoodieh Islamic University Tehran North Iran,

who contributed to this research.

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www.tropicalplantresearch.com 90Received: 22 March 2015 Published online: 30 June 2015

ISSN (E): 23491183ISSN (P): 23499265

2(2): 90100, 2015

Research article

Stem gall of Michelia champacaL. (Magnoliaceae) induced by

Podothripssp.: Identification, histochemical and

phytochemical studies

Monnanda Somaiah Nalini1*, K. E. Shilpa

1and Sekharappa Basavarajappa

2

1Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore570 006, Karnataka, India

2Department of Studies in Zoology, University of Mysore, Manasagangotri, Mysore570 006, Karnataka, India

*Corresponding Author:[email protected] [Accepted: 18 June 2015]

Abstract: Galls are organized structures of plant tissues induced by the insects. The present study

deals with the identification of gall-maker on the stem/twigs of Michelia champaca. The gall-

maker was identified as belonging to the order Thysanoptera, and was confirmed as the genusPodothrips. Histochemical staining was carried out in different portions of gall as well as healthy

twigs for the presence of alkaloids, fats, starch, tannins and proteins. The staining varied in

different tissues. The galls at different development stages (young and mature) were tested for the

presence of total phenolic as well as carbohydrate content. The results revealed high phenolic

content in young galls where as low amount was found in a mature gall tissue, however, a 2.3-fold

difference was noted between the two. Very high concentration of carbohydrate content was

detected in young gall tissues. The gall serves both as a shelter and a food source, to the gall-

maker. The inner walls of the galls are moist and are usually rich in phenolics and carbohydrates.

Our work reports for the first time Podothripssp. as gall-makers. Further studies on the ecology

of the insect would relate its occurrence on other host species and distribution.

Keywords:Gall -Michelia champaca- Histochemistry - Phenolics - Carbohydrate.

[Cite as:Nalini MS, Shilpa KE & Basavarajappa S (2015) Stem gall ofMichelia champacaL. (Magnoliaceae)

induced by Podothripssp.: Identification, histochemical and phytochemical studies. Tropical Plant Research

2(2): 90100]

INTRODUCTION

Michelia champaca L. is a medium to big sized tree with glossy leaves and yellow or orange flowers of the

family Magnoliaceae, native to the tropical and sub-tropical South and Southeast Asia, including Southern

China. The flower locally known as sampige or champa has great significance in Hindu mythology and has a

number of cosmetic, medicinal and economic uses. Fresh flowers are extracted into perfumes and medicinal

products which are used as cure for coughs and rheumatism. Cosmetic products such as Joy, Jadore and Dior

containM. champacafragrant extracts as ingredients in their composition (Armiyanti et al.2010).Michelia champaca is affected by biotic and abiotic factors. Among the biotic factors affecting the plants is

the presence of galls on the plant parts. Galls are abnormal growths on plants and are better understood as a suite

of adaptations to the inducing insect (Raman 2003). They are highly regulated growth manifestations on plants,

modified, symmetrical, natural-plant structures that are the outcome of messages from the inducing insects.

These structures develop as an extension of the host-plant phenotype (Raman 2012). Galls result from the

feeding of living organisms, such as insects, mites, and or mechanical stimulus. The attack of each species

results in a distinctive deformity on leaves, twigs or stem of a host plant. Such response also increases the

plants production of growth hormones such as auxins, cytokinins and gibberellins (Caron 2004).

Thrips are one class of well-known gall-forming insects, and belong to the order Thysanoptera. Thrips are

involved in several types of host relationship with plants, and their presence have been documented

(Ananthakrishnan & Raman 1979). Thrips (order-Thysanoptera) are tiny, slender insects with fringed wings.

They are very small, yellow, brown or black, slender insects ranging from 1/16 to 1/8 inch in length. Adults and


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larval thrips feed using a punch and suck technique. Their life cycle includes an egg stage, two larval instars,

two pupal stages, and an adult stage (Buss 2011).

Gall tissues are known to be sources of enzymes, phytochemical compounds such as alkaloids, fats, tannins,

proteins, starch, phenolics, carbohydrates etc. These accumulate in the tissues during gall development. Plant

phenolic compounds play an important role at different levels during plant-microbe interactions. A completely

different action of plant phenolic compounds lies in the defense of plants against pathogen attack (Vereecke et

al. 1997). Histochemistry is the branch of histology dealing with the identification of chemical components of

cells and tissues. Starch deposition occurs widely in the plant body, in the seeds, the parenchyma of the

secondary vascular tissue in the stem. Starch and protein are the principal ergastic substance of the protoplast.

Tannin is the heterogeneous group of phenol derivatives, usually related to glucosides. Fats are widely

distributed in the plant body and they probably occur in small amount of every plant cell. Many woody plants

contain medicinally important secondary products (Marmit & Sharma 2008) which can be analyzed using

staining techniques.

Various types of insect-plant galls exist in nature. Several plant species are reported to possess galls, yet the

occurrence of these in plants is to be documented. So far, no plant galls have been documented from Michelia

species. Since galls on the stem of Micheliawas first noted in the Manasagangotri Campus, we undertook the

study to document and identify the gall-inducer and analyse the phytochemical and histochemical differencesbetween the healthy and galled tissue portions of the stem.


Collection of the sample and study area

Healthy and galled twigs of Michelia champaca L., were collected from the plants growing in

Manasagangotri campus (12o18' N & 76

o12' E), Mysore, during the month of September to December, 2011.

The galled portions were excised from the stem with a metallic plier. The galls were separated according to their

size, as young and mature and photographed.

Identification of the gall-maker from the stem/twigs of Michelia champaca

Various stages of fresh gall materials such as the young and the mature (old) were collected. The collected

tissue was mounted on the clean glass slide and was kept on the field of Steriosome (Leica EZ4, MC234813,China). The gall tissue was dissected by using clean micro dissection needles and observed for the presence of

the gall-maker. Different stages of insects were photographed by using different magnifications, 835X. These

were considered for the documentation studies.

Histochemical comparison between gall and normal tissues

Free hand cut sections of young twigs ofMichelia champacawere taken by using clean and sharp blade and

floated in water contained in petriplates. Using pointed brush, fine transparent sections were selected and

immersed and placed in a cleaned glass slide and respective stains were added drop wise and covered with a

clean cover slip. Excess of stain was removed using a tissue paper and observed for the localization of viz.-

starch, tannins, proteins, fats and alkaloids under the microscope at various magnifications (4x, 10x and 40x)

and images were documented and compared.

Preparation of reagents

Reagents were prepared for histochemical studies according to the procedure of Momin & Kadam (2011).

Starch:0.3 g of iodine and 1.5 g of potassium iodide were dissolved in 100 ml of distilled water. A drop of the

solution was added on the section, left for some time and washed water and observed under microscope.

Protein:Saturated aqueous solution of picric acid is an excellent precipitating agent for protein, staining them

an intense yellow. The sections were allowed to react with the reagent for 24 h. The sections were washed

with 60% alcohol and few drops of aqueous FeCl3were added.

Tannins:Sections were treated with dilute acidic ferric chloride solution (0.5% to 1% of FeCl 3 in 0.1 N HCl),

mounted in clove oil and observed under microscope for the presence of tannins.

Fats:0.5 g of Sudan III and or Sudan IV is dissolved in 100 ml of 70% alcohol. Sections were immersed in the

stain for 20 min, rinsed quickly with 50% alcohol and mounted in glycerine for observations.

Alkaloids: Wagners reagent: One gram of iodine and 2 g of potassium iodide were dissolved in 50 ml of

distilled water.


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Estimation of total phenolic and carbohydrate content in the gall sample

Preparation of the extract: One gram each of young and mature gall samples were finely ground for few

minutes with 10 ml of 70% alcohol by using clean mortar and pestle. The extracts were taken in small

Eppendroff tubes and labelled. It was centrifuged at 5000 rpm for 10 min in a Spinwin centrifuge

(Tarsons, India). The supernatant was used for the estimation of total phenolic content.

Estimation of total phenolic content: The total phenolic content in the gall samples were determined by the

Folin-Ciocalteau (FC) method using Gallic acid as a standard (Volluri et al.2011) with slight modifications.

Different concentration of extracts (50250 g.ml-1) was mixed with 1.0 ml of Folin-Ciocalteau reagent (1:1

dilution). 2.0 ml of Sodium carbonate (20%, w/v) was added and shaken well. The reaction mixture was

incubated for 45 min under dark. After the specified incubation period the absorbance was read at 765 nm in

UV/Vis Spectrophotometer (T 60, TTL Technologies, India). The absorbance of standard as well as the

samples was plotted. The amount of total phenolics in the samples was expressed in terms of Gallic acid

equivalents (g ml-1GAE).

Estimation of total carbohydrate content: The total soluble carbohydrate content in the aqueous extracts of gall

tissues was determined using the phenol sulphuric acid method. Glucose was used as the standard

(Sadashivam & Manickum 2008). The aqueous extracts of the gall was obtained by boiling the known

amount of gall tissue (1 g.ml-1). The filtrate was used for the estimation of carbohydrates. A workingsolution was prepared by using different concentrations (1 mg.ml-1) of standard (525 g). The samples were

aliquoted (50250 l). The volumes of aliquots were made 500 l with distilled water. A blank was set with

500 l using distilled water.One ml of liquefied phenol solution (5%) was added to all tubes followed by the

addition of 2.0 ml of concentrated sulphuric acid immediately and shaken well. The reaction mixture was

incubated for 30 min in dark place.The absorbance of the standard as well as the samples were read at 490

nm using Spectrophotometer.The absorbance values and the amount of total carbohydrate in the different

samples was calculated and represented.

RESULTS

Identification of the gall on stem/twigs of Michelia champaca L.

Morphological observations: Gall onM. champacaoccurred on the main stem axis, as well as twigs, two feet

above the ground (Fig. 1B). The gall during initial development stages (young) appeared as snow-white out

growths (0.6 cm in diameter). They were formed at regular distance both on the stem as well as the twig

(Fig. 1C), as they matured, an increase in size (2.02.5 cm in diameter) was observed and later coalesced to

form large sub-spherical structures. One of the important observations made during the gall development is

the change from the initial snow-white colour (Fig. 1C) to slightly blackish structure at maturity (Fig. 1D).

Observation of mature galls by Steriotrinocular microscopy showed the presence of small black glandular

hairs filled with exudates (Figs. 1E & F).

Identification of the gall-maker: The gall-maker on the stem/ twigs ofM. champacawas identified as belonging

to the order-Thysanoptera, which include Thrips. Upon periodical and successive observations it was

confirmed as the genus Podothrips. On the basis of the characters, and the availability of various stages of

development (Fig. 2), only one insect chamber was observed from the under surface of the gall. The bodysize observed was 0.51.0 mm. The systematic position is as follows: Class: Insecta; Order Thysanoptera;

Family: Phlaeothripidae; Genus:Podothrips.

Histochemical studies: A comparison of section of galled, gall normal and healthy portion showed remarkable

differences anatomically (Table 1). In healthy portion, a conspicuous pith and sub-spherical outline of

vascular bundles with the larger bundles alternating with the smaller bundles was observed (Fig. 3A-a). In

gall normal, pith is conspicuous and the outline of vascular bundles is sub-spherical, the larger bundles

alternate with smaller bundles (Fig. 3A-b). The section resembles the healthy plant. In galled portion,

inconspicuous pith, radial growth of vascular bundles and conspicuous sclerenchyma cap was observed (Fig.

3A-c). The following are the results of the tests.

Alkaloids:A golden yellow colour revealed the presence of alkaloids (Figs. 3B-ac). In healthy tissue,

sclerenchyma cap and metaxylem and vascular bundles and entire pith region were lightly stained. In gall

normal stem tissue, an intense stain was observed in sclerenchyma cap and cells of metaxylem showed less


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intensity of stain and in pith region only centre portion were stained. In gall stem tissue, the sclerenchyma

cap, vascular bundles, medullary rays and metaxylem cells showed the high intensity of stain and outer cells

of the pith region was stained less intensely.

Figure 1. Galls onM. champaca L.: A,Healthy plant; B,Galls observed two feet above the ground level; C,Stem and twigsshowing galls; D,Close up indicating slightly blackish colour; E, Increased size with black glandular hairs filled with

exudates; F,Close up.


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Figure 2.Developmental stages of the gall-maker: A,Healthy plant; B,Infected plant; C,galls on twig; D,Single gall; E,

Dorsal view of gall; F,Ventral view of gall; G,Young thrip; H,Adult thrip.

Fats:Blue colour indicated the presence of fat. In healthy tissue, high intensity stain was observed in

sclerenchyma cap, vascular bundles and medullary rays and less intensity in pith cells, cortex and epidermis.

In gall normal stem tissue, sclerenchyma cap is continuous and forming a ring like structure. In these

vascular bundles, sclerenchyma cap and cortex showed the high intensity and less in pith cells. In galled

stem tissue, sclerenchyma cap was discontinuous. Sclerenchyma cap, medullary rays and vascular bundles,showed the high intensity of stain, very less in pith regions (Figs. 3C-ac).


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Tannins:Blue colour indicated the presence of tannins. In healthy tissue, in xylem elements dark stain was

observed, high intensity of stain was observed in the sclerenchyma cap, vascular bundle and medullary rays.

The pith region showed very high intensity of stain. In gall normal stem tissue, the sclerenchyma cap,

vascular bundle and medullary rays showed the high intensity of stain and in pith region showed the very

high stain. In galled tissue, except the sclerenchyma cap all cells showed the high intense stain (Figs. 3D-a

c).

Table 1. Histochemical differences in the healthy and galled twig ofM. champacaL.

Histochemical

testsColor

Histological components stained

Healthy twig portion Galled twig portion

Alkaloids Goldenyellow

Sclerenchyma cap, vascular bundles &entire pith (+)

Sclerenchyma cap, vascular bundles,medullary rays (+++) & outer pith (+)

Fats Cobaltblue

Sclerenchyma cap ( Continuous ring),vascular bundles, medullary rays

(+++); pith cells, cortex, epidermis (+)

Discontinuous sclerenchymatous cap,vascular bundles, medullary rays (+++)

Tannins Prussianblue

Sclerenchyma cap, vascular bundles,

medullary rays & pith region (+++);

Sclerenchyma cap (+); vascular bundles,medullary rays & pith region (+++)

Starch Blue Cortex, medullary rays & pith (+++); Sclerenchyma cap, metaxylem elements& pith region (+++); cortex (+)

Proteins Intenseyellow

Sclerenchyma cap, vascular bundles,

medullary rays & pith region(+)

Sclerenchyma cap, vascular bundles,medullary rays (+++);pith region (+)

Note:+, indicates light staining; +++, indicates moderate staining; +++, indicates intense staining.

Starch:Blue colour indicated the presence of starch, in healthy tissue, high intensity starch was observed in

cortex, medullary rays and pith cells, pith is conspicuous (Fig. 3E-a). In gall normal tissue, cortex,

metaxylem cells, medullary rays showed high intensity of stain. In pith region very high intensity of stain

was observed (Fig. 3E-b). In galled tissue, sclerenchyma cap, metaxylem elements and pith region showed

the intense stain, more in vascular region and less in cortex (Fig. 3E-c).

Protein:An intense yellow colour indicated the presence of proteins; almost all the tissue showed intensestaining. In healthy tissue, less intensity of stain was observed in the sclerenchyma cap and very less in all

parts of the sections (Fig. 3F-a). In gall normal tissue, high intensity of stain was observed in sclerenchyma

cap and metaxylem cells and less in vascular bundles and medullary rays and pith region showed less

intensity (Fig. 3F-b). In galled tissue, sclerenchyma cap, metaxylem elements, medullary rays and vascular

bundles showed the very high intensity and less intensity in the pith region (Fig. 3F-c).

Total phenolic and total soluble carbohydrate contents

Total phenolics:The phenolic contents of young and mature gall tissue having differences in the growth stages

were compared with Gallic acid as standard. In young and mature galls, the phenolic content of 240 g.ml-1

GAE and 225 g.ml-1

GAE were observed (Fig. 4). Both did not differ much in their phenolic contents.

However, a 2.3-fold difference was noted, when expressed in terms of weight of the gall tissue.

Total soluble carbohydrates: The total soluble carbohydrate content of young and mature gall tissues showing

much difference in the growth stages were compared with glucose as standard. The total soluble

carbohydrate was 9.4 g.ml-1 in young galls, whereas the mature gall contained 0.5 g.ml -1 (Fig. 5).

However, an 18.8-fold increase was noted in the concentration in the young galls when compared to mature

galls.

DISCUSSIONS

Insect galls are organized structures of plant tissue which form in response to either feeding by the gall-

maker (phytophagy) or egg-laying (oviposition) on plant tissue. There are number of galls types induced by the

insects on leaf, stem, twig, bud or flower and fruit. In the present investigation, galls on Michelia champaca

(Magnoliaceae) were noticed for the first time on the stem/twigs. So far, a stipular scar forming a ring around

the stem (node) inMagnolia virginianahas been reported to be caused by a psyllid, Trioza magnolia(Ashmead)

(Hall 2009).


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Figure 3. A, Healthy sections stained with safranin stain; Histochemical tests for: B,Alkaloids; C,Proteins; D,Fats; E,Tannins; F,Starch (a,Healthy portion; b,Gall normal portion; c,Galled portion).


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Figure 4. Determination of total phenolic content in the gall tissues of M. champaca; Ethanolic extracts of young and mature

gall tissues were prepared (1g 10 ml-1). Concentrations ranging from 50250 g.ml-1 were tested for the total phenolic

content by Folin-Ciocalteu method using gallic acid as standard. The absorbance of the samples as well as the standard was

read at 765 nm and the values of the samples were calculated from the standard graph and represented in terms of g.ml-1

GAE.

Figure 5. Determination of total soluble carbohydrates in gall tissues ofM. champaca: The total carbohydrate content of theaqueous extracts of young and mature galls were determined by phenol-sulphuric acid method using glucose as standard.

Concentrations ranging from 50250 g.ml-1were tested for the carbohydrate content. The absorbance of the samples and

the standard was read at 490 nm and the values of the samples were calculated from the standard graph and represented.

Galls are made up of cells that are more numerous or larger than the normal plant cell or plant organs whose

growth and development have been altered into unusual shapes. An insect gall is initiated because of the plants

response to the insects egg laying (oviposition) or presence of the egg, and/or feeding stimulation by the larva

(phytophagy). Plant cells are usually modified and enlarged, the plant tissue surrounds the egg or larva, and the

gall protects and feeds the gall-inducer. Galls are located on rapidly growing plant parts-on catkins, seeds,

flowers, petioles, branches and stems; most occur on leaves and buds. Some galls are single-chambered

(monothalamous) and contain only one gall-maker, and others are multi-chambered (polythalamous) which

contain many gall-makers (Buss 2011).

Gall onM. champacaoccurred on the main stem axis, as well as lateral twigs, two feet above the ground.The gall during initial development stages (young) appeared as snow white out growths (0.6 cm in diameter).


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They were formed at regular distance and as they matured galls increased in size (2.02.5 cm diameter) and

coalesced to form large sub-spherical structure. Gopinathan & Suresh (1985) worked on the solid, indehiscent

galls on the stem of Pongamia glabra induced by an undescribed agromyzid (Diptera). They reported, that

young galls (815 612 mm) were relatively soft and green and with maturation, they became harder and

developed white patches and is directly proportional to the number of larvae residing inside. Mature galls

showed generally, a single gall chamber extending along the pith region.

The gall-inducer was identified as belonging to the genus Podothripsof the order- Thysanoptera, family

Phlaeothripidae. Only a single insect chamber was observed from the under surface of the gall. The body size

observed was 0.51.0 mm. The genus Podothripsare reported for the first time as gall-inducers. Furthermore,

thrips as gall-makers have been reported on plant species (Raman & Ananthakrishnan 1989). Thrips such as

Gynaikothrips uzeli induced galls on the leaves of Ficus bengalensis (Borbon 2011). Gynaikothips ficorum

caused leaf gall onFicus laevigata(Santis 1980). Although, we have documented the evidence of this genus as

a gall-inducer, worldwide Podothrips are reported to cause huge losses to crops such as chilli (Scirtothrips

dorsalis), which is an important pest of crops in tropical and subtropical regions in Florida (Ludwig & Bogsan

2007).

A comparison of sections of galled, gall normal and healthy portion showed remarkable differences

anatomically. The following tests were conducted to localize viz., alkaloids, fats, starch, tannins and proteins intissues. In healthy portion, pith was conspicuous and the outline of vascular bundles was sub-spherical, the

larger bundles alternated with the smaller bundles. In gall normal, pith is conspicuous and the outline of vascular

bundles is sub-spherical, the larger bundles alternate with smaller bundles. The section resembles the healthy

plant. In the galled portion, inconspicuous pith, radial growth of vascular bundles and conspicuous

sclerenchyma cap was observed.

In the present study alkaloids, tannins, fat, proteins and starch were detected in various tissues and differed

considerably in healthy and galled portions of the sections. Our observations are supported by the studies of

Gopinathan & Suresh (1985) who worked on solid, indehiscent galls on the stem of Pongamia glabrainduced

by an undescribed agromyzid(Diptera). In the histochemical studies, maximum concentrations of proteins were

observed in the nutritive cells of medullary region and phloem cells. Starch deposits occurred in the pith cells,

medullary rays in normal stem sections, whereas in gall portions they were located in the outer region of pith ofthe nutritive zone. Tannins were present less in gall tissues, while they were totally absent in normal stem.

Kumar & Mathur (2009) reported the histochemical localizations in st

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