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WSN 52 (2016) 181-194 EISSN 2392-2192

Galls on scholaris as air pollution indicator

Partha Talukdar1, Kaushiki Das2, Shrinjana Dhar2, Soumendra Nath Talapatra2 and Snehasikta Swarnakar3,* 1Department of , Srirampore College, University of Calcutta, William Carey Road, Hooghly, , 2Career Advancement Solutions, Maheshtala, – 700142, India 3Cancer Biology and Inflammatory Disorders Division, CSIR - Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata – 700032, India *E-mail mail address: [email protected] ; *Phone: +913324995759

ABSTRACT Air pollution arises mainly from automobiles and industries is well known fact. Monitoring and detection by instrument cannot be possible everywhere however, indication from by their alterations in morphology and anatomy may be a suitable easy screening measurement. The present study aims to detect morphological features with special reference to gall quantification and anatomy of leaves of R. Br., found in eastern Indian urban and suburban area that are exposed to vehicular emission. The results indicated alterations of leaf morphology along with length (L), breadth (B), L/B ratio and significantly increased (P < 0.001, 0.01 and 0.05) Air Pollution Index (API). It is concluded that vehicular emission can be monitored as an early indication through increased API in A. scholaris. Further research would be needed in relation to secondary metabolites alteration, biochemical and genetic parameters to know pollutant susceptibility as an indicator. In addition, anatomical abnormalities in gall formation as well as numbers were also pronounced in leaves exposed to various load of air pollution.

Keywords: Automobile air pollution; gall formation; bioindicator plant; leaf morphology and anatomy; Air Pollution Index (API); Alstonia scholaris

World Scientific News 52 (2016) 181-194

1. INTRODUCTION

Air pollutants generate from different activities viz. automobiles, fugitive and stack, burning of solid wastes etc. Beside these, automobiles cause both primary and secondary air pollutants in India, which has already been reported by regulatory authorities (CPCB, 2009; Citizen’s Report, 2011) and several researchers (Joshi and Swami, 2007; Diwvedi et al., 2008). It has been established that air pollution impact on the plant species worldwide in relation to plant-environment interactions, since the are more sensitive as well as resistant species compare to other organisms. The abnormalities are including plant external morphology, anatomy, physiology and/or biochemical profiles indicate about the polluted environment. The air pollutants response varies in plants as species to species and also in terms of different types, reaction mechanisms, concentration and exposure time. The pollutants when enter into the plants then react before being removed or absorbed and may lead to accumulation, chemical transformation and incorporation into the metabolism system. In these processes, some plants are injured while others show minimal effects or no impacts (Choudhury and Banerjee, 2009). Among other trees, Devil tree, Alstonia scholaris, R. Br. () is an evergreen, tropical tree with white funnel-shaped and milky sap and grows to height of 30-40 m found in most of parts of India. Among medicinal properties, A. scholaris is also used as an avenue tree because this plant is prescribed in greenbelt and easy to manage in polluted areas, requires less water and bioindicator plant (Muhammad et al., 2014; Mandal Biswas et. al. 2014). The plant species under greenbelt can effectively be prescribed as air pollutants prevention as resistant (tolerant) and sensitive or responding (Warren, 1973; Singh and Rao, 1983; Tiwari and Tiwari, 2006). The gall formation rate is higher when plant species are susceptible to air pollutants and induce the nitrogen content in the leaves then improve the herbivore palatability. Furlan et al. (2004) have hypothesized in Tibouchina pulchra (Cham.) Cogn. (Melastomataceae), a native species of the Brazilian Atlantic forest has pollution- resistant capacity. According to them, when stress caused by air pollutants, the levels of secondary metabolites decreased, while the contents of nitrogen increased, due to a partial blockage of primary metabolism participated in the of protein and other nitrogenous compounds synthesis. They found the reduced amounts of secondary metabolites and the increased amounts of foliar nitrogen, have found the potential to enhance herbivore foliar damage. Generally the leaf galls of A. scholaris induced by a bug, Pauropsylla tuberculata crawf., which is an insect (class Psyllidae, order homoptera) as reported by researchers (Hodkinson, 1984; Mandal Biswas et al., 2014). The research work has revealed that formation of galls induced by homoptera insects, correlated with the feeding habit and predominantly inhabited in leaf galls (Meyer, 1987). The insects are known to extract nutrients from the phloem, xylem or non-conducting plant cell (Arya et. al., 1975). The insect activates a perturbation in growth mechanisms and alters the cellular differentiation processes of the leaf in the host plant, modifying the plant architecture as per its advantage (Raman, 2007). Many studies as bioindicator plants showing visible leaf damages, morphology, anatomy and biochemical changes related to air pollution internationally (Middleton et al., 1956; Bull and Mansfield, 1974; Husen et al., 1999; Naveed et al., 2010; Seyyed and

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Koochak 2011) as well as nationally (Tiwary et al., 2008; Saquib et al., 2010; Deepalakshmi, 2013; Nandy et al., 2014). The previous studies have emphasized on alteration of physico- chemical parameters by air pollution. On the other hand, different studies carried out on foliar galls morphology, anatomy and biochemistry after insect (Pauropsylla tuberculata) infection and deposition their eggs (Albert et al., 2011; Mandal Biswas et al., 2014). The present study attempts to know the morphological and anatomical deformities along with galls quantification on leaves of A. scholaris found near roadside in eastern India.

2. MATERIALS AND METHODS 2. 1. Study area The study areas were selected as per vehicular loads. The study was carried out at 4 sampling stations viz. (i) low vehicular load (LVL) as control area, (ii) moderate vehicular load (MVL), high vehicular load (HVL) and (iii) heavy vehicular load (HeVL) as experimental area. These four sampling stations were selected on the basis of low, moderate, high and heavy traffic density along with vehicular movement as per visualization. The plant species was selected Alstonia scholaris R. Br. growing near roadside of above mentioned area because gall formations are more common in this species. The affected leaf shape was determined by the study of shape as length (L), breadth (B) and L/B ratio and anatomy of leaves along with galls. It was also studied number of galls per leaf. All the leaves randomly selected from 5 trees of above-mentioned area.

2. 2. Area of Leaves The 10 leaves were collected randomly from per tree of above-mentioned area. Individual leaf was cleaned properly in running water and soaked with blotting paper. The area of leaves especially L/B (Length / Breadth) ratio of leaf (in cm), was measured manually.

2. 3. Galls quantification The 10 leaves per tree were collected randomly. Individual leaf was cleaned properly in running water and soaked with blotting paper. The quantification of galls was done as per leaf by manual counting. The gall formation rate was evaluated by an index, namely Air Pollution Index (API) was postulated by following formula:

Air Pollution Index (API) = No. of galls per centimeter2 x Whole leaf L/B ratio

2. 4. Anatomical observation The 10 leaves per tree were collected randomly. Individual leaf was cleaned properly in running water and soaked with blotting paper. The anatomical observation was done under bright field microscope (Olympus) with a 40x magnification.

2. 5. Statistical analysis All the mean values of data were determined statistically significant differences between experimental and control leaf samples for morphological features by using Student’s

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t-test (P < 0.05 level). The statistical analyses were done by using software (Microsoft Ver. 8.1, Excel 2013 with add on statistical tool pack).

3. RESULTS AND DISCUSSION

The present results clearly indicate that vehicular load brought significant changes in foliar morphology, numbers of gall formation and anatomy of leaves and gall in A. scholaris (Table 1 and Fig. 1, 2, 3, 4 and 6). Fig 1a and b show significant changes in green colour indicating high chlorophyll content in normal leaves in comparison to excessively affected bugs having yellowish green. According to Albert et al. (2011), chlorophyll content is varied from new gall leaves to mature gall leaves when compared to ungalled or less galled leaves. In all experimental sites such as MVL, HVL and HeVL, the extra growth and sometimes reduction pattern were significantly (P < 0.001, 0.01 or 0.05) observed when compared to control site (LVL) for L, B and L/B ratio as morphological features (Table 1). For the parameter L, significantly decreased values (P < 0.001) were observed for both area viz. MVL and HVL but the value was also decreased in HeVL area at a significant level of P < 0.05 when compared to LVL. In case of the parameter B, the values for both area viz. MVL and HVL were decreased significantly (P < 0.01) but for HeVL decreased at a significant level of P < 0.05 when compared to LVL. The values for L/B ratio were also decreased significantly for all study area compared to control area. The important morphological parameter as gall formation onto leaf lamina were showed an increasing trend at a significant level of P<0.001. It was found the gall quantity with an increased value of 6 fold in MVL, 7 fold in HVL and 9 fold in HeVL in comparison to LVL (Table 1). Several researchers in previous findings have documented the morphological deformities. According to them, reduction in leaf area due to particulates pollution and particulates along with other air pollutants such as O3, SO2 and NOx, PAN have more damaging effect on leaves (Joshi and Swami 2007; Tiwari et al., 2008; Deepalakshmi, 2013; Nandy et al., 2014), which supports the present study.

Table 1. Measurement of the shape of leaves due to the presence of galls

Sl. No. Area Parameters (n = 10; M ± SD)

Gall quantity L (cm) B (cm) L/B (cm) (nos.) 1. LVL 23.8 ± 1.60 4.57 ± 0.53 5.20 ± 0.29 2.40 ± 2.17

2. MVL 12.69 ± 3.82* 3.51 ± 0.81** 3.55 ± 0.34* 12.30 ± 5.56*

3. HVL 15.84 ± 1.88* 3.94 ± 0.52** 4.02 ± 0.32* 14.3 ± 6.57*

4. HeVL 21.01 ± 4.74*** 3.97 ± 0.71*** 3.65 ± 0.49* 18.7 ± 7.04* *P < 0.001; **P < 0.01; ***P < 0.05

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The visible injuries such as cholorosis, pigmentation, necrosis etc. in leaves were not found in all the study area but pathogenesis may alter the colour of the leaves as green to yellowish green (Fig. 1). All images in different types of external gall shapes viz. newly formed, matured and perforated after releasing of bugs were depicted in Fig. 2 a, b and c. Moreover, visible injuries are potent indication of air pollution and the parts and/or whole plant showed susceptibility to individual and/or combination of air pollutants (Saquib et al., 2010; Deepalakshmi, 2013; Nandy et al., 2014).

(a) (b) Fig. 1. Morphological features along with galls (a) control leaf and (b) experimental leaf

(a)

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(b)

(c) Fig. 2. Morphological features in experimental area: (a) newly formed galls, (b) matured galls and (c) perforated galls after releasing bugs

The leaves of A. scholaris exist in whorls of seven in numbers. The leaf dorsal surface is called as abaxial and the ventral surface is known as adaxial. In the present study, it was observed that the leaves of experimental area were more yellowish green and normal green in control area. The infected leaves were observed growth reduction with crumple in shape (Fig. 3a, b, c and d). It was also recorded that galls formation were found onto both surface of the leaves while in some experimental area only from abaxial side, which is supported by Albert et al. (2011). The study of whorls of A. scholaris is also an important parameter in morphology.

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(a) Adaxial view and maximum numbers of newly formed galls in experimental leaves

(b) Abaxial view and maximum numbers of newly formed galls in experimental leaves

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(c) Adaxial view and less numbers of newly formed galls in control leaves

(d) Abaxial view and less numbers of newly formed galls in control leaves

Fig. 3. Morphological features of leaf of experimental (a and b) and control area (c and d)

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The tree, A. scholaris were found at a height of 40-45 feet (Fig. 5a). The present study of gall formation on the leaves of Alstonia scholaris indicates that leaves below 10-15 feet height were showed maximum numbers of galls while beyond 15 feet height no galls observed near roadside of MVL, HVL and HeVL areas in comparison to LVL (Fig. 5a, b and b). It is hypothesized that gaseous pollutants from vehicles may be diffused within 20foot height. Several researchers have been documented that air pollutants cause changes in morphology, anatomical features, growth rate, biochemical, physiological profiles and also injuries on the leaves of trees (Iqbal et al., 1996; Ghosh et al., 1998; Viskari et al., 2000; Oksanen and Holopainen, 2001; Peeters, 2002; Furlan et al., 2004; Nandy et al., 2014).

(b) No galls formation beyond 15 feet height

(c) Newly formed galls within (a) A. scholaris tree (40-45 feet height) 10-15 feet height

Fig. 4. Gall formation a particular height of tree.

It is also noted that API value was significantly higher (P < 0.001) as per gall quantification in the experimental area compared to control area as represented by bar diagram (Fig. 5). In our study, API values were found an increasing trend at significant level in HVL and HeVL area but increased without significant level in MVL when compared to LVL area. This is the first report that API can be an early detector of air pollutants to A. scholaris. In addition, APTI (Air Pollution Tolerance Index) has already been documented by other researchers, in which total chlorophyll, ascorbic acid, leaf extract pH and relative water

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content were examined to know sensitivity of the trees towards air pollution (Singh and Rao 1983; Choudhury and Banerjee, 2009; Tripathi et al., 2009). In support, tree species, A. scholaris has exhibited low APTI value and established a sensitive species to air pollutants exposure. Altogether, our data revealed that increased numbers of gall formation and higher API values in the leaves of A. scholaris may be suitable indicator of air pollution.

Fig. 5. Graphical representation of Air Pollution Index (API) versus vehicular load in different study area (n = 10; M ± SD; *P < 0.001)

The anatomical features were found deformities both dorsal and ventral surface of leaves along with the internal structure of gall. It was observed deformed epidermis and palisade tissue when compared to control area and deformed gall shape (Fig 6a, b, c and d). In the control leaf it was found cells are tubular, compact and covered with thick cuticle in upper epidermis while papillae with thick cuticle in lower epidermis. It was also observed hyperplasia in the palisade tissue. The present anatomical abnormality due to numerous gall formations on experimental leaves is found closely similar work reported by Albert et al. (2011). Moreover, it is suggested to visualize deformities under electron microscope, which can be resulted in detailing cellular features.

(a) (b) (c) (d)

Fig. 6. Anatomical observations (in cross section): (a) control leaf, (b) leaf with early gall, (c) deformed epidermis and palisade tissue in leaf having early formed gall and (d) deformed shape of matured gall

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Air pollution can also induce qualitative and quantitative changes in secondary metabolite composition (Lea et al., 1996; Zobel, 1996; Kanoun et al., 2001; Lopanen et al., 2001). These secondary metabolites help as protection against insect herbivores, pests and pathogens. According to Dicke, 2000; Gatehouse, 2002 and Furlan et al., 2004, abnormal abiotic factors such as air pollutants have capability to change the structure and/or quantity of secondary metabolites in leaves. In this context, air pollution can also inhibit carbon capture capacity in plant and decrease nitrogen:carbon ratio, ultimately reduced the level of carbon- based secondary compounds while enhanced the level of nitrogen-based compounds (Jones and Coleman, 1991; Hamilton et al., 2001; Furlan et al., 2004). Another important phenomenon e.g. fibers and lignin production require carbon that hamper due to the alterations of secondary metabolites (Furlan et al., 2004). The present results are contradictory that insect herbivory induced gall formation only by chemical stimuli in A. scholaris as observed by Albert et al. (2011) and Mandal Biswas et al. (2014). However, there may be alterations of secondary metabolites by air pollutants where insect herbivory affect the leaves of tree, A. scholaris, subsequently abnormalities observed as reported by Gatehouse, (2002) and Furlan et al., (2004). It is further an interesting observation by Holopainen and Oksanen (1995) that arboreal insects are suitable indicator of air pollution because these species found in maximum numbers for host plant herbivory and parasitism can be easy due to increase level of nitrogen content. It was documented that herbivorous insects with different feeding habits, respond to stress-enhanced changes in their host trees with different intensity (Larsson, 1989). According to Larsson, gall forming insects adapt to air pollution stress in host plant and are already considered as air pollution indicator. It was established that gall-forming aphids such as Sacchiphantes sp., Adelges sp., etc. have been reported with higher population densities in zones of heavy air pollution (Ranft, 1968; Pfeffer, 1978).

4. CONCLUSION

In conclusion, this study is an assessment of host-plant herbivory and the gall formation onto leaf, which is a can be a suitable indicator to know API index as a primary test of air pollution. According to Central Pollution Control Board (CPCB), A. scholaris is a suitable tree for greenbelt to mitigate air pollution but this tree species has documented as a sensitive species among other plants in relation to air pollution (Singh and Rao 1983; Tripathi et al., 2009). On the other hand, gall forming insect species is also found on the leaves of trees during stress-induced condition, for example, air pollution (Holopainen and Oksanen, 1995). Further researches should be needed to understand phytochemicals content alteration, biochemical and genetic damages in A. scholaris along with abnormalities of gall forming insects in vehicular loaded area.

Acknowledgement

The authors convey their thanks to Botany Hons. 2nd year students Souvik and Souptik, Department of Botany, Bangabasi and Sreerampore College, University of Calcutta, for help during sample collection for the present study. Also thankful to Mr. Binayak Pal, Sr. Technical Officer, CSIR-Indian Institute of Chemical Biology, Kolkata for photography of all images in present manuscript.

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( Received 05 July 2016; accepted 19 July 2016 )

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