Studies on Aerobiological Status of Panaji and Surrounding Areas

July 2010

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STUDIES ON AEROBIOLOGICAL STATUS OF

PANAJI AND SURROUNDING AREAS

Thesis Submitted to THE GOA UNIVERSITY for the award of the degree of

Doctor of Plhilosophy in Botany

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By

Mrs. Mangala P Prabhudesai, M.Sc.

Department of Botany Goa University

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DECLARATION

As required by the University ordinance 0.19.8 (ii), I hereby declare that the Ph.D thesis titled

"STUDIES ON AEROBIOLOGICAL STATUS OF PANAJI AND SURROUNDING AREAS"

submitted to Goa University, forms an independent work carried out by me in the Department of Botany, Goa University, under the supervision of Dr. K.G. Hiremath, Head, Department of Botany, Dhempe College of Arts and Science, Miramar-Goa and Prof. D.J. Bhat, Professor, Department of Botany, Goa University. The thesis has not formed previously the basis for the award of any degree, diploma, associateship or other similar titles.

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Mrs. Mangala P Prabhudesai

Counter-signed by:

K.G. Hiremath (Guide)

(Co-Guide)

CERTIFICATE

We certify that the thesis titled "STUDIES ON AEROBIOLOGICAL STATUS OF PANAJI AND SURROUNDING AREAS" submitted by Mrs. Mangala P Prabhudesai is a record of research work done by her during the period from July 2005 to January 2010 when she worked under our supervision. The thesis has not formed the basis for the award of any degree, diploma, or associateship to Mrs. Mangala P Prabhudesai.

We affirm that the thesis submitted by Mrs. Mangala P Prabhudesai incorporates the independent research work carried out by her under our supervision.

Guide:

Co- Guide:

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(Dr. K.G. Hiremath)

Acknowledgements

I wish to express my sincere gratitude to my guide Dr. K.G. Hiremath, Head, Department of Botany, Dhempe College of Arts and Science, Miramar, Goa and my co-guide Prof. D.J. Bhat for their constant encouragement and valuable guidance throughout the course of my work. I am thankful to Prof. P.K. Sharma, Head, Department of Botany, Goa University, for his constant support and Prof. M.K. Jamarthanam, Prof. B.F. Rodrigues, Dr. Vijaya Kerkar, Dr. S. Krishnan and Dr. N. Kamat for their valuable suggestions and support during the course of this work.

I wish to thank Dr. S.V. Deshpande, Principal, Dr. Y. Modassir, Vice-Principal, and the Management, Dhempe College of Arts and Science, Miramar, Panaji, Goa, for the facilities granted during this work. I wish to thank my colleagues at Dhempe College, Dr. S.R. Ganihar, Dr. Purnima Ghadi, Mr. S.J. Godse, Dr. P.N. Pangu, Ms. Manjiri Kelkar and Mr. G.I. Huklceri for their constant encouragement and motivation during the work.

I am grateful to Prof. S. N. Agashe for his advice during the initial phase of this study, at a time when I was unaware of many intricacies of this subject.

I am grateful to Dr. Omkar Singh, former Chief Conservator of Forests, Government of Goa for granting me permission to conduct studies at Mollem and Cotigao wildlife sancutaries.

Dr. V.S. Korikantimath, former Director of ICAR, Old Goa, and Dr. K. V. Singh, Director, India Meteorology Department, Panaji, Goa, are thanked for sampling facilities and weather data provided during the course of sampling.

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Thanks to Dr. Seema Kamat, Mr. Nitin Sawant, Mr. Paresh Parab, Mr. Deepak Tandel, Mrs.

Sonia Phaldesai, and Mr. Purshottam Jathar for logistic help during the sampling period.

I am indebted to Dr. Vivek Prabhudesai, Ponda, for sharing his vast experience on allergy from a medical perspective.

I am thankful to Dr. Puja Gawas Saldialker, Dr. Pratibha Jalmi, Ms. Sarita Yadav and Mr. Ashish Prabhugaoker for their valuable help.

I sincerely thank my brother, Mr. Rajendra Bhandari, a renowned expert in the mechanincal engineering fraternity, for fabricating an effective and simplistic spore-trap for use in this study, as per the recommendations and suggestions of several aerbiologists across India.

I fondly acknowledge the support from my sons, Shantanu and Sumant, during the course of my research work.

I am grateful to all those who have been directly and indirectly responsible for the successful completion of this work.

(Mangala Prabhudesai)

Contents

Chapter 1 Introduction 1

Chapter 2 Literature Survey . 5

Chapter 3 Material and Methods .24

Chapter 4 Results ... 34

Chapter 5 Discussion 106

— Chapter 6 Summary 128

References 133

Published papers 153

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List of Tables

Table 1- Location of study sites and sampling frequencies. 31 Table 2 - Seasonal Density and diversity of pollen: Miramar 2005-2006 60 Table 3 - Seasonal Density and diversity of pollen: Miramar 2006-2007 62 Table 4 - Seasonal Density and diversity of spores: Miramar 2005 - 2006 64 Table 5 - Seasonal Density and diversity of spores: Miramar 2006 - 2007 67 Table 6 - Seasonal Density and diversity of pollen: Altinho 2005-2006 70 Table 7 - Seasonal Density and diversity of pollen: Altinho 2006-2007 72 Table 8 - Seasonal Density and diversity of spores: Altinho 2005-2006 74 Table 9 - Seasonal Density and diversity of spores: Altinho 2006-2007 77

Table 10 - Seasonal Density of pollen: ICAR 2005-2006 80

Table 11 - Seasonal Density of pollen: ICAR 2006-2007 82

Table 12 - Seasonal Density of spores: ICAR 2005-2006 84

Table 13 - Seasonal Density of spores: ICAR 2006-2007 87

Table 14 - Seasonal Density of pollen: Farmagudi 2005-2006 89 Table 15 - Seasonal Density of pollen: Farmagudi 2006-2007 89 Table 16 — Seasonal Density of Spores: Farmagudi 2005- 2006 90 Table 17 — Seasonal Density of Spores: Farmagudi 2006- 2007 91 Table 18 — Seasonal Density of Pollen: Cotigao 2005- 2006 92 Table 19 — Seasonal Density of Pollen: Cotigao 2006- 2007 92 Table 20 - Seasonal Density of Spores: Cotigao 2005- 2006 93 Table 21 - Seasonal Density of Spores: Cotigao 2006- 2007 93

Table 22 - Seasonal Density of Pollen: Mollem 2005- 2006 94

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Table 23 - Seasonal Density of Pollen: Mollem 2006- 2007 94

Table 24 - Seasonal Density of Spores: Mollem 2005- 2006 95

Table 25 - Seasonal Density of Spores: Mollem 2006- 2007 95

Table 26 - Seasonal Density of Pollen: Mandrem 2005- 2006 96 Table 27 - Seasonal Density of Pollen: Mandrem 2006- 2007 96 Table 28 - Seasonal Density of Spores: Mandrem 2005- 2006 97 Table 29 - Seasonal Density of Spores: Mandrem 2006- 2007 97

Table 30 - Seasonal Density of Pollen: Margao 2005- 2006 98

Table 31 - Seasonal Density of Pollen: Margao 2006- 2007 98

Table 32 - Seasonal Density of Spores: Margao 2005- 2006 99

Table 33 - Seasonal Density of Spores: Margao 2006- 2007 99

Table 34 — Weather parameters and pollen and spore count for the period of 2005-2006 100 Table 35 — Weather parameters and pollen and spore count for the period of 2006-2007 101 Table 36 — Correlation of pollen and spores with respect to various weather parameters 102 Table 37 — Representation of allergic reactions of ten patients sampled at the clinic in Ponda,

Goa 124

Table 38 - Pollen Calendar 126

Table 39 - Spore Calendar 127

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List of Figures

Fig. 1 - Geographical location of the sites where the study was carried out 25 Fig. 2 - A small lump of glycerine jelly was kept on the slide, heated on a spirit lamp flame. 28 Fig. 3 - The slide was coated with a thin film of glycerine jelly. 28 Fig. 4 - Observation of exposed slides under the microscope for identifying pollen and spores 29

Fig. 5 — Spore-trap showing the mounting of slide 29

Fig. 6 — Slide fixture on the spore-trap 30

Fig. 7 - Pollen and Spores encountered during the study 103

Fig. 8 - Spores encountered during the study 104

Fig. 9 - Spores encountered during the study 105

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Chapter 1

Introduction

Louis Pasteur in 1860 proved through his classical experiments that air is the carrier of many common germs. Since then, several workers have analysed the air, its chemical and biological components and their impacts (Blackley, 1873; Airy, 1874).

`Aerobiology' is a scientific, multidisciplinary field aimed at understanding the presence, transport and distribution of microorganisms and other biologically significant materials in the air and its consequences on the living as well as non-living world (Edmonds and Benninghoof,

1973). It is particularly concerned with airborne microorganisms, especially their sources, take off, dispersal, deposition and effects on other organisms - a process termed as aerobiological pathway (Edmonds, 1979). Aerobiological studies deal with the diagnosis of composite living or non-living parts in the air, their morphology, physiology, viability, sampling, concentration, diurnal and seasonal patterns, phenology, emission, transportation, dispersion, pollination, pollinosis and a host of other subjects (Agashe, 2006). Aerobiology has also been defined as the study of microbial population in the atmosphere, now designated as airspora. According to Purohit and Ranjan (2005), aerobiology deals with the presence of aerial biomass, such as pollen grains, fungal spores, trichomes, microscopic fragments of members of algae as well as insects, protozoans, crustaceans and other unidentified entities.

The dominant biomass which contribute to the load of air are pollen grains of various higher plants belonging to families such as Amaranthaceae, Asteraceae, Fabaceae, Poaceae and Solanaceae, and spores of fungal genera such as Alternaria, Aspergillus, Bispora, Cladosporium, Curvularia, Drechschera, Helminthosporium, Nigrospora and Pyricularia. Stem and leaf trichomes, microscopic fragments (filaments of blue-green-algae) of members of Cyanophyta and tiny insects add to the air-load. The ramifications of aerobiology are wide and varied

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covering diverse sectors of human pursuit. The areas of interest may be classified into primary sector (agriculture), secondary sector (industry) and tertiary sector which includes forestry, health, energy, environment, tourism and socio-economic components like eco-development and criminology (Nair, 1994). Aerobiological studies have also been employed for the control of illicit drug adulteration by identifying and analyzing the airborne fungal spores present in drugs such as heroin and cocaine. The presence of fungal conidia in dry drug powder arises from contamination during the period extending from its synthesis to packing. Certain analysis revealed that the fungal contamination in brown heroin was significantly high. It has been proved by Dominguez (1994) that pollen grains and fungal spores aid in the construction of events in forensic sciences.

The term "aerobiology" was coined in the 1930s while analyzing fungal spores, pollen grains and bacteria in the atmosphere by F.C. Meier who was a distinguished plant pathologist working in the Department of Agriculture, United States of America. Preliminary aerobiological work and its applications in alleviation of human sufferings from environmental pollution date back to the period of the Vedas in 3000 B.C. (Agashe, 2006). Ancient literature such as the Vedas and Agnihotra has references of polluted air and its purification methods (Tilak, 1982).

Sreeramulu (1986) opined that research on airspora especially that of crop fields, has much to contribute in the understanding of diseases on crops and vegetation. The primary objective of aerobiological studies is to detect, determine and monitor the occurrence of pollens and spores and their distribution in the atmosphere. Aerobiology has now become an interdisciplinary subject of great significance and application in different fields such as ecology, medicine, pathology, agriculture, forestry and meteorology:The primary activity of an aerobiologist is

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therefore to construct a pollen/spore calendar of the region which will be useful to allergologists and patients suffering from allergy.

Gregory (1952) suggested the term "airspora" to describe fungal and pollen flora suspended in the air. Subsequently, other biological particulates such as plant fragments, insects and insect parts, protozoan cysts, seeds etc. were included in the study of aerobiology (Agashe, 2006). In the recent past, the scope of aerobiology has been widened to incorporate different types of biological particles in the air (airspora), viz. virus, bacteria, microalgae, fungi, lichen fragments, tiny seeds, protozoan cysts, insect and insect parts and spiders. Agashe (2006) pointed out that the pollen constitutes a small part of aeroplankton or airspora in the atmosphere. The most frequent and dominant particles of biological origin in the air are propagules of microorganisms such as spores of fungi. Particles when dispersed in air are termed as 'aerosols'.

Algae, leaf hairs, seeds, plant fragments and volatile material including scents and terpenes also occur in aerosols. The atmosphere may also contain other particulates including bushfire ash, industrial ash and fly ash from incomplete fuel combustion.

The aerobiological process comprises of five recognizable issues with reference to airspora, viz. source, release, dispersion, deposition and impact (Frinking et al., 1977).

Throughout the development of aerobiology, the emphasis of studies has successively involved all these issues (Lacey, 1994). Nilsson (1981) divided the aerobiological studies into intramural (indoors) and extramural (outdoors).

Aerobiological investigations have been carried out by a number of researchers in India.

These include Padmanabhan et al. (1952), Kongar et al. (1958), Kasliwal et al. (1959), Sreeramulu and Seshavataram (1962), Sengupta and Chattopadhyay (1968), Mehrotra and

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Cladius (1968), Sreeramulu and Vittal (1971), Sharma and Sinha (1973), Reddi (1974, 1976), and Pande (1976). Establishment of the Aerobiological Society of India in 1980 is a testimony of the importance gained for such information in various service sectors, especially in atmospheric pollution, health and agriculture.

Present study:

There is very little information available on aerobiological status of west coast of India, especially of Goa, Karnataka and Maharastra. Hence this work was taken up. The present study, carried out for duration of two years from June 2005 to May 2007, is an effort to collate baseline data on the airspora of Goa, qualitatively and quantitatively. The pollens, fungal spora and other extraneous materials present in the air were sampled out at Panaji and a few neighbouring localities in Goa, at different periodicities.

The following objectives have been projected in the preparation of this thesis.

1. Determination of the biological composition of airspora of Panaji and neighbouring regions in Goa.

2. Determining the density, diversity and seasonal variations of airspora.

3. Analysis of the data based on statistical indicators (such as diversity index, species richness and evenness).

4. Comparison of the data obtained in this work with hitherto aerobiological studies done in India and abroad.

5. Documenting the allergenic airspora and its comparison with the findings in other regions of India.

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Chapter 2

Literature Survey

The earliest observation of microorganisms in the air was made by Hippocrates (ca. 460 BC — ca. 370 BC). He believed that some of the diseases occur through inhalation of contaminated air. Lucretius in 55 B.C. held a view that particles were carried by wind. In the beginning of 19th century it was clear that pollen of higher plants and spores of fungi, bryophytes and pteridophytes were liberated into the air and transported by wind. Several Indian workers have assessed the quantum of pollen discharge into the atmosphere (Khandelwal and Mittre,

1973; Agnihotri and Singh, 1975; Reddi, 1976; Mondal and Mandal, 1998).

Aerobiological studies by the International Biological Program (IBP) have led to the establishment of International Aerobiology Association (I.A.A.) in 1974 at Hague, Netherlands.

It greatly helped to promulgate new approaches to aerobiology. The major function of I.A.A. is the promotion, in the largest possible sense, of aerobiology as a scientific discipline. Several countries in Europe have daily news bulletins about the incidence of pollen in the air which serves as a useful information to the allergenic patients.

Currently abiotic particulates or gases affecting living organisms have been included in the concept of aerobiology by International Biological Programme (IBP) in 1964. Major aerobiological research centers in India are located in Srinagar, New Delhi, Shillong, Lucknow, Jaipur, Gwalior, Bhopal, Kolkata, Aurangabad, Visakapatnam, Chennai, Bangalore, Mysore and Thiruvananthapuram (Agashe, 2006).

The first report on systematic aerobiological study in India was by Cunningham (1873) who carried out aerobiological survey over Presidency jail premises of Culcutta. His work was published in the book "Microscopic Examination of Air" (Cunningham, 1873). He attempted to relate the incidence of cholera and other fevers using an aeroconiscope in Culcutta jails. About

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half a century after Cunningham's pioneering work, aerobiological studies in India were once again conunenced by plant pathologists such as Mehta (1952) and Padmanabhan et al. (1952).

Systematic investigations on airborne pollen were initiated early at different localities in India. Kasliwal et al. (1955, 1959) studied the atmospheric pollen grains of Jaipur. Lakhanpal and Nair (1958) surveyed atmospheric pollen at Lucknow. Sreeramulu (1959) carried out systematic and intensive studies on aeromycology at Visakhapatnam using Hirst volumetric spore trap. Shivpuri et al. (1960) and Dua and Shivpuri (1962) studied the aerobiology of Delhi.

Subsequently two new centers, one at Aurangabad and another in Mysore came into existence.

The former was initiated by S.T. Tilak in 1966 and the latter was started by A. Ramalingam in 1965. Aerobiological investigations were commenced during 1970s at Bose Institute, Culcutta by S. Chanda and in Bangalore by S.N. Agashe in 1973. Three others centres, one each at Allahabad, Nagpur and Gorakhpur, came into existence more or less at the same time in the 1970s. Aeromycological studies at Madras were started by B.P.R. Vittal using volumetric samplers in 1976. Many centres came up during the 1980s which include Gwalior, Jabalpur, Santi Nilcetan, Manipur, Bodh Gaya, Gulbarga, and Trivandrum.

Systematic aerobiological investigation, especially of airborne pollen, was initiated at Patel Chest Institute, Delhi in 1957 by Shivpuri and his co-workers. Sreeramulu (1960) studied the spore content of Darjeeling. Nair (1963) surveyed airborne pollen at Vellore. Gupta and Singh (1965) prepared a pollination calendar of allergenic plants of Bilcaner and &tied out aerial survey of pollen and fungi. Baruah and Chetia (1966) studied airspora of Guwahati from allergy point of view. Sreeramulu (1967) reviewed the aerobiological status of India. Talde (1969) studied the suspended airspora over banana fields in Parbhani, Maharastra. Subba Reddi

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(1970) carried out a comparative survey of atmospheric pollen and fungal spores at two places twenty miles apart. The aerobiological study of Aurangabad caves was carried out by Tilak and Kulkarni (1972). Pande (1976) studied the airspora over several agricultural fields at Nanded, Maharastra. Millins et al. (1976) identified the sources and incidence of airborne Aspergillus fumigates.

Shukla and Mishra (1978) made a survey of pollen at Kanpur, U.P. and prepared a pollen calendar. Appanna (1980) prepared a pollination calendar of potentially allergic pollen- producing plants of Vijaywada, Andhra Pradesh. Mandal and Chanda (1981) carried out aeropalynological sampling in Kolkata city. Dosi and Kulkarni (1981) have carried out a preliminary survey of the airspora of Mumbai. Gaur and Kasana (1981) studied aerobiology of Modi Nagar, Gujarat. Singh (1981, 1983) conducted an airborne pollen survey and preparation of pollen calendar in Shillong. Satpute et al. (1983) contributed to the aerobiology of Shillong and studies related to the seasonal variations of atmospheric pollen and fungal spores. Ghai (1984) studied the airspora of the suburbs of Mumbai. Ithare (1984) studied airspora with special reference to fungal spores at Rani Durgawati Vishwavidyalaya Campus in Jabalpur, M.P.

Agashe and Chatterjee (1987) carried out aeropalynological survey at different altitudes using aircraft sampling method, in Bangalore. Yeragi and Sasikumar (1985) undertook an extensive survey of airspora of the industrially polluted Ambernath and Ulhasnagar, the distant suburbs of Mumbai. Nair et al. (1986) surveyed the airborne pollen, spores and other materials of India.

Bhat and Rajasab (1988) studied the incidenc,e of airborne fungal spores at two different sites in Gulbarga. Malik et al. (1990) investigated the concentration of pollen allergens at human height.

Srivastava and Shukla (1990) carried out investigation of airspora of Balrampur city, West Bengal. Vaidya (1990) studied airspora at Aurangabad and its relevance with environmental 7

parameters. Lacey et al. (1990) studied the airborne microorganisms associated with domestic waste composting. Singh (1991) published a summary of three decades of aerobiological research in North East India. Jain and Datta (1992) listed pollen grains in the airspora of Gwalior. Chaturvedi et al. (1992) wrote an account of incidence of grass pollen in Indian environment. Agashe and Sudha (1995) studied circadian periodicity of fungal spores in Bangalore city. Spilcshma et al. (1995) studied the trends and fluctuations in annual quantities and the starting date of birch (Betula) pollen in Europe. Arora (1995) carried out aerobiological survey of Churu District of Rajasthan. Reddi and Reddi (1996) performed aeromycological survey of Vilcarabad. Satheesh et al. (1997) studied the airborne pollen incidence in relation to season and vegetation at Kodailcanal, Tamil Nadu. Rapiejko et al. (1998) evaluated pollen count at different heights and distance. Satheesh Kumar and Vittal (1998) carried out a preliminary survey of airborne pollen in Madras City. Khadelwal (2001) surveyed airspora of Lucknow.

Mishra et al. (2002) studied circadian periodicity of airspora in different seasons in Jabalpur.

George and Varma (2002) studied seasonal and diurnal variations of airborne fungal spores in Jabalpur. Devi et al. (2002) studied the airspora of setni—urban areas of Guwahati city. Sahney and Chaurasia (2004, 2008) have monitored the incidence and density of grass pollen in the atmosphere of Allahabad. Aeropalynological studies at Pulwama District of Kashmir have been carried out by Mudasir et al. (2006). Tiwari et al. (2006) have compiled a pollen calendar of Raipur and also identified the pollen grains of allergenic nature. Pandey et al. (2006) carried out aeropalynological survey of Banda District, U.P. An aerobiological survey of Guwahati was conducted by Devi (2007). Jyothi and Bhagyalakshmi (2007) conducted aeropalynological studies of exhibition grounds in Hyderabad. Hazarika et al. (2007) presented a report of the aerobiological studies carried out at Assam. Das and Gupta - Bhattacharya (2007) have shown

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the relationship between airborne culturable fungal flora of an agricultural farm in West Bengal and meteorological factors.

Aeromycological studies in India have progressed along three different lines (Vittal, 2005): (1) Outdoor aeromycology, (2) Indoor aeromycology and (3) Air mycoflora of crop fields. Studies on mycoflora of indoor air were so far relatively few in India compared to outdoor studies. The indoor environment included houses, hospital wards, libraries, poultry houses, cow sheds, bakeries, grocery shops, go-downs, leather store-houses, etc.

Detailed studies on aerobiology of diseases of groundnut, cereals, pulses, vegetables, oilseeds and cash crops are available. The investigations have indicated a close correlation between meteorological factors, growth stages of crop and spore load in the atmosphere. Spore level within and above crop fields has been monitored and it has been found that the concentration within the crop was maximum when compared to higher elevations.

Aeromycological investigations were carried out in the caves of Ajanta and Ellora. The significant role played by the airborne biodeteriogens in deleterious effects of cave paintings has been well documented (Nair, 1960; Dua and Shivpuri, 1962).

Vittal and Glory (1985) analysed airborne fimgal spores of a library in India.

Murdhankar and Pandey (1991) worked on aerobiological and epidemiological appttach to groundnut rust. Singh and Dorycanta (1992) have carried out aerobiological survey over a maize field in Senapati District in Manipur. Ramachander Rao (1993) studied the epidemiology of airborne conidia of Alternaria on sunflower. Chawda and Rajasab (1994) studied day to day variations in the incidence of Alternaria porri conidia over a purple blotch infected onion field.

Raha and Bhattacharya (1997) evaluated aeromycoflora of residential areas of two distinct 9

biozones of West Bengal. Verma et al. (1997) observed the diversified fungal spore concentration in the poultry environment. Lohare and Kareppa (2000) investigated the aeromycoflora over Soybean field at Udgir (Maharashtra) for two kharif seasons. Chakraborty et al. (2000) carried out an indoor and outdoor aeromycological survey in Burdwan. Mitakakis and Guest (2001) prepared a fungal spore calendar for the atmosphere of Melbourne, Australia.

Chakraborty et al. (2003) evaluated aeromycoflora of an agricultural farm in West Bengal, India.

Kasprzyk and Worek (2006) published data on airborne fungal spores in urban and rural environments in Poland. Tiwari and Saluja (2007) have presented seasonal variation of aeromycoflora of

at Raipur (C.G.). The aeromycological survey over

Mentha arvensis

plantation at Raipur (C.G.) was carried out by Singh and Tiwari (2007). Pund et al. (2007) published aeromicrobiota at different sites of Amaravati City (Maharashtra). Dhavale and Reddi (2007) carried out the survey of the atmospheric microbiota over sugarcane field at Ahmedpur (Maharashtra). Survey of fungal spora in the industrial units of Guwahati was carried out by Devi et al. (2007). Singh (2006) studied aerobiology, epidemiology and forecasting of fungal diseases found in certain crops of North-Eastern states of India. Debnath and Baruah (2007) monitored the incidence of airborne mycoflora of a tea field at Jorhat district, Assam.

Mohture et al. (2007) studied aeromycoflora of a semi-urban area of Wanjra, Nagpur.

Chakrabarti et al. (2007) carried out aeromycological studies over an agricultural field near Kolkata, West Bengal. A quantitative and qualitative assessment of the indoor mycoflora of a school building was carried out by Majumdar et al. (2007, 2008). Kalkar and Tatte (2007) investigated aeromycoflora of indoor environment in hospitals of Nagpur. Investigations on fungal spora of a playground in Pimpri, Chinchwad area was carried out by Ahire et al. (2007).

Bharati et al. (2007) studied the fungal airspora of Maria coalfields. Sahney and Purwar (2007)

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surveyed airborne fungi at Allahabad. Majumdar and Ghara (2008) studied the indoor aeromycoflora of school buildings in Kolkata. Cholke and Mahajan (2008) carried out studies of aeromycoflora inside poultry shed. Debnath and Baruah (2008) evaluated seasonal variation of air mycoflora over tea plantation in Jorhat district, Assam.

Pollen and Climate:

Climatic changes influence vegetative growth, reproductive cycle and in turn, pollen counts. Relative humidity, temperature, wind speed and rainfall are the most important factors in daily variations of pollen grains (Emberlin et al., 2004). The warm dry season stimulates flowering in some tree taxa and aids dehiscence (Solomon, 1979). Smart et al. (1979) published comprehensive information on the relation between weather parameters and pollen counts in Australia. According to their findings, grass pollen incidence was greatest on days with high maximum temperature and reduced on days of high humidity and low temperature. Jarai- Komlodi and Madzihradzky (1994) are of the opinion that the pollen dispersion may be influenced by a number of internal (physiological, phyto-ontogenical and antho-biological) and external (meteorological) factors, varying from year to year. McDonald (1980) opined that heavy rains may delay the stamens to open and may suppress pollen dispersion. Lacey (1981) has studied the temperature effect on hygroscopic movements and water rupture of pollen grains that help it to liberate easily into the atmosphere. According to McCartney (1994) the dispersal of aerobiological particles is the result of complex interactions between the biological and physical factors. The effect of biological factors may differ between species and location, but the physical mechanism of dispersal is essentially the same for all particles. Rebeiro et al. (2003) found that air-borne pollen concentration significantly correlated with certain meteorological parameters.

Pollen concentration was positively correlated with temperature and wind direction and negatively correlated with rainfall and number of rainy days. Rodriguez-Rajo et al. (2003) published the correlation between pollen content in the atmosphere of Lugo (NW Spain) and weather parameters. A positive correlation was seen with temperature, hours of sun and wind speed and negative correlation with rainfall and relative humidity. Prospero et al. (2005), after studying the response to strong El Nino, suggest that long- range transport of microorganisms might be particularly responsive to climate variability. Mesa et al. (2003) analysed that rainfall and maximum temperature are important factors controlling the magnitude of grass-pollen season in southern Spain and United Kingdom. Mandal et al. (2006) reported that the variation of pollen load showed a positive correlation with all the meteorological parameters except mean maximum temperature and relative humidity, where it showed a negative correlation. Mansour and Abdel (2005) published data of their aerobiological studies at Giza district, Egypt, in which they mention that rainfall and relative humidity reduce airborne pollen counts, whereas temperature has the greatest influence on daily pollen count because more vapours in the atmosphere may hinder the mobility of airborne pollen and also, rainfall washes out pollen from the atmosphere. Clot (2003), after overviewing twenty years data in airborne pollen at Neuchatel (Switzerland) confirmed the airborne pollen to be a sensitive indicator of climate change. Munshi (1994) prepared a pollen calendar of Kashmir University campus and opined that no clear relationship could be established between wind velocity and pollen concentration. Burt and Rutter (1994) concluded from their studies that there was no definite relationship between spore catch and weather parameters like relative humidity and wind speed. McCartney (1994) has reported that wind has an enormous capacity to disperse material. Air borne pollen grains generally travel short distances, however, when they are blown into the upper strata of the 12

atmosphere, pollen grains travel long distances. Pollen count is reduced after precipitation (Shivanna, 2003). Rainfall and relative humidity reduce airborne pollen count whereas temperature has the greatest influence on daily pollen count because more vapours in the atmosphere may hinder the mobility of the airborne pollen and rain washes out pollen from the atmosphere. Moreover heavy rainfall delays the dehiscence of stamens and may suppress pollen dispersion. Burt and John (1994) showed that there is no relationship between spore catches and relative humidity and also that there is no significant correlation between conidial counts and wind speed. Magda et al. (1994) have observed the influence of a number of internal factors such as physiological, phyto-ontogenical, anthobiological, and external, such as meteorological factors varying from year to year to influence the pollen dispersion. The long-range transport of microorganisms might be particularly responsive to climate variability in general.

Temperature affects the hygroscopic movement and water rupture of pollen grains that help to liberate pollen easily to the atmosphere (Lacey, 1981). Prospero et al. (2005) suggested that microorganisms and dust concentration were unusually great in 1997, possibly in response to the strong El Nino effect. The range of transport of microorganisms might be particularly responsive to climate variability in general. Jan et al. (2003) analysed that rainfall and high temperature are important factors controlling the magnitude of the grass pollen season in both southern Spain and UK and that the strength and direction of the influence exerted by these variables changes with geographical location and time. Rebeiro et al. (2003) showed a significant correlation between airborne pollen concentration and certain meteorological parameters. Pollen concentration positively correlated with temperature and wind direction and negatively correlated with rainfall and number of rainy days. McCartney (1994) observed that the dispersal of aerobiological particles is the results of complex inter-reactions between biological and

1 3

physical factors. The effects of biological factors may differ between species and location but the physical mechanisms of dispersal are essentially the same for all particles. Griffin (2003) observed that dust particles serve as a vessel for global dispersion of bacteria and fungi. Dust borne microorganisms may play a significant role in the ecology and health of down wind ecosystem. Rodriguez-Rajo et al. (2003) correlated annual pollen with hours of sun, temperature, wind speed and relative humidity. The results were positive with hours of sun and wind speed and negative with rainfall and relative humidity. It may be noted from the above literature survey that the effect of biological, meteorological and anthobiological factors are not uniform but have shown a great variability in their response.

The ultimate aim of an aerobiologist is to compile a pollen/spore calendar, which will be useful to allergologists and patients suffering from allergy. The main objective of the continuous air sampling is to get qualitative and day-to-day variations in the concentrations of different pollen and fungal types. Such data enables compilation of the pollen calendar, which depicts the duration and concentration of various pollen types in the atmosphere. Pollen calendars compiled by aerobiologists provide knowledge of the occurrence and concentration of allergenic pollen, which is of great help to clinicians for proper diagnosis. Pollen calendars should be compiled and updated every year, because the magnitude and quality of annual pollen load in the atmosphere can vary significantly (Sudha, 1992). There can be significant variation in the atmospheric pollen even between two successive years. This aspect was highlighted by Agashe and Abraham (1990). A pollen calendar of Chennai for the year 2001 has been compiled and published by B.P.R. Vittal, Uday Prakash and Bhuvaneshwari of the Center of Advanced Studies in Botany, University of Madras, Chennai.

14

Pollen calendars have been prepared by several workers in India for various places. As a pre-requisite to the evaluation of allergenicity by an allergologist, the lcnowedge of the pollen calendar of the local region is essential. Pollen calendar was prepared by Karla and Dumbrey (1957) for Poona; Sanghvi et al. (1957) for Rajasthan; Subba Reddi (1970) for Visakhapatnam;

Khandelwal and Mittre (1973) for Lucknow; Jha et al. (1975) for Varanasi; Deshpande and Chitaley (1976), Chanda and Mandal (1977) for Kalyani in West Bengal; Tripathi et al. (1978) for Bhopal; Bora and Baruah (1980) for Guwahati; Appanna (1980) for Vijayawada, Andhra Pradesh; Gaur and Kasana (1981) for Modi Nagar; Jain and Das (1981) for Gwalior; Singh and Babu (1982) for Delhi, Tripathi et al. (1982), Nayar and Ramanujam (1989) for Secunderabad.

Pollen calendar of Bangalore city was compiled for two years, 1982-83 and 1983-84 (Agashe and Abraham, 1990), which showed the count and relative abundance of 15 major pollen types.

Aerobiologists have also prepared pollen calendars for various sites outside India.

Atmospheric survey conducted in France and a comparative study by Monpellier and Font- Romen provided pollen calendars for Alder, Pinus, Rumex, Cupressaceae, Poaceae and Urticaceae. The pollen calendar for Switzerland was provided by Leuschner (1974) at Switzerland and at Germany by Stix (1974). Pollen calendar for Huddinge, Sweden was formulated on a five years survey by Nilsson and Pahnberg-Gotthard (1982). Jarai—Komlodi and Madzihradzky (1994) prepared a five years pollen calendar at Hungary (1989-1993).

Pollen calendars have also been published for Stockhohn (Nilsson and Praglowski, 1974;

Engstorm and Nilsson, 1979; Juhlin-Dannfelt, 1984), Alexandria (Ghazly and Fawzy, 1988) and Turin (Caramiello et al., 1989).

15

Other Uses of Aerobiology:

The ramifications of aerobiological studies are wide and varied covering diverse sectors of human pursuit. The areas of interest may be classified into primary sector (agricukure), secondary sector (industry) and the tertiary sector including, forestry, health, energy, environment, tourism and socio-economic component like eco-development and criminology (Nair, 1994). Long-term aerobiological survey of an area can be used in the construction of statistical models for prediction of the start and intensity of pollen season. (Bringfelt, 1982). It has been proved that pollen grains and spores can aid in the reconstruction of events in forensic sciences. Aerobiological studies have been employed for the control of illicit drug adulteration by identifying and analysing the airborne fungal spores present in drugs, such as, heroin and cocaine. The presence of fungal conidia in dry powder arise from contamination during the period extending from its synthesis to its packing .Analysis revealed that the fungal contamination in brown heroin was significantly more than in white heroin and cocaine (Dominguez-Vilches, 1994).

Aerobiology and allergy:

Air contains an array of biological particles of different shapes and sizes. These bioparticles (pollen grains, fungal and algal spores and fragments, dust and dust mites, animal proteins) when inhaled cause Type I respiratory disorders in certain genetically predisposed individuals. Such substances are known as inhalant allergens. The term allergy was first introduced in 1906 by Freiherr van Pirquet, an Austrian physician, to describe any abnormal reaction on the inunune system. Allergy is defined as altered and accelerated reaction of a person to a second and subsequent exposure to a substance to which his body has already become 16

sensitized by a previous exposure. Aerobiological studies in relation to allergy, has a great relevance as the problem of allergy is assuming alarming proportions all over the world. All pollen grains occurring in the atmosphere do not cause allergy. The pollen grains which cause allergy have to fulfill certain basic requirements stated by Thommen (1931), such as:

1. They should contain an excitant to cause allergy.

2. They should be wind pollinated.

3. They should be buoyant enough to be carried away easily by wind.

4. They should be produced in huge amounts.

5. The plant producing such pollen grains must be widely and abundantly distributed.

Van-Halment (1607) was the first to report an annually occurring asthma due to the pollen. Floyer (1726) reported the first case of fungal sensitivity with regard to allergy.

Significance of pollen as allergen was started in 1766, when Koelreuter reported dissemination of pollen by wind. Bostock (1819) for the first time suspected pollen as a cause of hay fever.

Elliotstan (1831) pointed out the positive allergenicity of Poaceae and Amaranthus. The firSt complete description of hay fever was given by Botok in 1819, but he incorrectly attributed it to the heat of the sununer. Ehrenberg (1872) published information on micro-organisms from atmospheric dust for the first time. Charles Blackley, who was also a sufferer of hay fever, noted that the disease was more prevalent in educated town dwellers who were exposed to high amounts of pollen. He noted that some army officers in India were afflicted with hay fever only where there was grass, or only when they came back to England in the summer and very seldom at high altitudes. He found that his own hay fever was better at sea side, but only when the wind was from the sea or when sailing in a boat far out to sea or after the hay was cut. In 1857 his 17

children brought some early flowering grasses into the house and caused an attack before the season, perhaps providing the final stimulus for his prolonged investigations.

In 1831 Elliotstan suggested that pollen of plants growing in meadows cause hay fever.

Blackley (1873) proved the validity of this view by self-inhaling Penicillium spores and producing acute asthma. He carried out the first bronchial provocation test. Dunbar (1903) demonstrated the proteinaceous nature of the allergenic factor of pollen grains and treated the patients with serum pollantin obtained by immunizing the horses with pollen extracts. Ever since the aerobiological work was initiated by F.C. Meier in 1936, extensive global progress has taken place with reference to allergy and has been reviewed by various workers from time to time. The airborne particulate matter of less than 10p diameter plays an important role along with allergen exposure to decrease pulmonary function in asthmatics (Gupta-Bhattacharya et al., 2007). An extensive study of many objectives was carried out in Europe and has been published in "Atlas of European allergenic pollen grains" edited by Charpin and Surinych (1974). Basett et al. (1978) have prepared a similar atlas for North Europe and Canada. Some examples of the health ailments caused are cold, hay fever, allergic bronchial asthma, allergic rhinitis and atopic dermatitis (Hyde, 1969; Stanley and Linskens, 1974; Knox, 1979; Leuschner, 1993; Agashe,

1994). Constance and Therese undertook pollen monitoring at Sydney, a city for Olympic Games in 2002, out of concern that pollen sensitive athletes may have significant problems with allergic symptoms triggered by pollen exposure. They found extremely high pollen count at the sites chosen for training and competition. The study of airborne pollen at the University of Rome (Caiola et al., 2002) indicated that the prevalent allergies were due to Graminae, Urticaceae and Oleaceae pollen.

18

In India, research on the identification of offending allergens in patients with bronchonasal disorders and their treatment by immunotherapy was initiated by D.N. Shivpuri in early sixties at V. P. Chest Institute, Delhi. Studies on aerobiology in relation to respiratory allergy in India were carried out by Kasliwal et al. (1955). Sanghvi et al. (1957) made an aeropalynological survey from allergy point of view at Rajasthan. Kasliwal and Solomon (1958) made a correlative study of pollen frequency along with respiratory allergy in Rajasthan.

Shivpuri et al. (1960) canied out skin, conjunctival, bronchial and other clinical and immunological tests with antigens extracted from spores of different species of fungi and pollen grains prevalent in Delhi area. Baruah and Chetia (1966) showed the relation between airspora and allergic human diseases. Sulemani and Gupta (1969) studied the role of causal allergens in 200 cases of bronchonasal allergy in Bikaner. Agarwal et al. (1969) studied the allergic fungal spores of Delhi. Chanda and Sarkar (1972) noted that the pollen grains of Greater Culcutta are a causative agent for respiratory allergy. Davies and Smith (1973) studied the airspora to forecast the start and severity of hay fever season. Jha et al. (1975) performed clinical tests with some airborne pollen and dust recovered from Varanasi area. Darke et al. (1976) diognised respiratory diseases of workers harvesting grains. Chanda and Ganguli (1976) studied the role and chemistry of some potential allergenic pollen as environmental pollutants in India. Shukla and Mislu -a (1978) made a survey of pollen at Kanpur and prepared a pollen calendar and also studied their allergenic significance. Sarpotdar and Rajmane (1978) studied the asthamogenicity of pollen of Parthenium hysterophorus. Tiwari (1978) produced results of hyposensitisation from 100 cases of nasobronchial allergy caused by local allergens in Bhopal. Shivpuri (1978) worked on the influence of cliinatic factors and the role of offending allergens that cause hypersensitivity in susceptible individuals. Prakash et al. (1978) worlced on clinical aspects of pollen allergy. Mittal 19

et al. (1978) reported the results of intradermal tests by using pollen antigens on some patients of nasobronchial allergy in Kanpur. Babu et al. (1979) noted a defmite correlation between the amount of pollen grains in the atmosphere and the incidence of allergy. Chaubal and Gadve (1979) studied aeropalynology and pollen allergens in Kolhapur. Vishwe (1979) observed the correlation of seasonal and perennial incidence of allergenic symptorns with the atmospheric pollen incidence and found Cassia tora pollen to give high positive skin reactions. Ajay Shankar (1979) observed that pollen grains of Brassica, Ricinus, Adathoda, Ageratum and Artemissia were clinically significant in Gwalior. Greveson (1979) has worked on fungi as a causative agent of allergic diseases. Appanna (1980) prepared a pollination calendar of potentially allergic pollen producing plants of Vijaywada, Andhra Pradesh. Acharya (1980) performed slcin test response to some inhalant allergens in patients of nasobronchial allergy from Andhra Pradesh Shivpuri (1980) identified clinically important pollen, fungal and insect allergens for nasobronchial allergy patients in India. D'Silva and Freitas (1981) have studied the role of aerial mycoflora of Mumbai in respiratory allergies. Tilalc et aL (1981) reported a number of patients hypersensitive to pollen antigens of some local plants, including Parthenium hysterophorus. Anand et al. (1981) reported the scope of aerobiological studies in immunotherapy. Mandal and Chanda (1981) performed clinical tests with the pollen of Cucurbita maxima and Lantana camara and found them to give significant positive reactions. Batabyal et al. (1985) observed the manifestation of atopic and non-atopic allergy induced by pollen of Chenopodium album. Rao et al. (1985) evaluated allergenic aeffects of Parthenium hysteroporus pollen. Chemical analysis of the pollen grains of Acacia auriculifortnis was performed by Agarwal et al. (1986). Davies et aL (1988) studied occupational asthma in tomato growers following an outbreak of the fungus Verticillium alboratum in the crop. Singh et aL (1988) studied the influence of climatic factors on airborne

20

pollen allergens. Detection of grass pollen allergy was done by Kundu et al. (1988). Sahay et al.

(2001) studied allergic reactions due to Aspergillus fumigatus. Santra et al. (1991) found pollen of Datura metel and Cocos nucifera to be allergenically potent. Saoji and Giri (1994) have worked on concentration of aeroallergenic fungal spores in intramural environments of Nagpur city. Vijay (1994) conducted advanced studies on mould allergens of Altemaria altemata.

Yasmeen and Saxena (1996) studied the aerial biopollutants of Aligarh. Katelaris et al. (2000) recorded daily pollen counts since 1994 as a part of Olympic Pollen Project to identify the risks associated with Olympic Games for allergic athletes. Adhilcari et al. (2000) assessed the fungal aerosol of a bakery with reference to allergic significance. Chauhan et al. (2004) reported allergenic significance of airborne fungi of Agra city. Verma and Jacob (2005) studied allergenicity of fungal spora in.the environment of cattle sheds at Jabalpur. Mir and Bhattacharya (2005) also found pollen of Cassia tora to be one of the major causes of respiratory allergy.

Airborne pollen is a good model to study the interrelation between air pollutants and respiratory allergic diseases. Mandal (2005) investigated the nature of aeroallergens from different parts of Durgapur, a highly industrial area of eastern India, in terms of their role as organic environmental pollutants. Saroja and Bhagyalakshmi (2005) have studied the impact of biological pollution on human health. Mandal et al. (2006) have identified the allergenic components of Peltophorum pterocatpum. Gupta-Bhattacharya et al. (2007) opined that the airborne particulate matter of less than 10m diameter play a important role along with allergen exposure to decrease pulmonary function in asthmatics.

Aerobiological work in Bangalore was initiated from 1973 because of sizable number of residents suffer from allergic manifestations. Reports from Asthama Research Society clahn that

1.31% of the population suffer from asthma. Studies by Agashe and Elizabeth (1990) show that a 21

number of cases of asthma in Bangalore, correlated to the peak season of Parthenium, Casuarina, Ricinus and Amaranth-Chenopod.

Meteorological Parameters:

Rainfall and relative humidity reduce airborne pollen counts whereas temperature has greatest influence on daily pollen count, because more vapours in the atmosphere may hinder the mobility of the airborne pollen and also heavy rainfall washes out pollen from the atmosphere.

More,over, heavy rainfall delays the dehiscence of the stamens and may supress pollen dispersion. However, Burt and Rutter (1994) showed that there was no relationship between spore catch and relative humidity. Also, that there is no significant correlation between conidial counts and wind speed. Magda and Medzihradsky (1994) have observed the influence of a number of internal factors, such as physiological, phytoontogenical, anthobiological and external, such as meteorological factors, varying from year to year to influence the pollen count.

McCartney (1994) observed that the dispersal of aerobiological particles is the result of complex interactions between biological and physical factors.

Dispersion:

Temperature affects hygroscopic movements and water rupture of pollen grains that help to liberate pollen easily to the atmosphere (Lacey, 1981). Prospero et al. (2005) suggested that microorganisms and dust concentration were unusually high in 1997, possibly in response to El Nino. The long range transport of microorganisms might be particularly responsive to climatic variability in general. Mesa et al. (2003) found that rainfall and high temperature are important factors controlling the magnitude of grass pollen season in both southern Spain and United

22

Kindom, and that strength and direction of the influence exerted by these variables differs with geographical location and time.

Rebeiro et al. (2003) showed a significant correlation between airborne pollen concentration and certain meteorological parameters. Pollen concentration positively correlated with temperature and wind direction and negatively correlated with rainfall and number of rainy days. The effects of biological factors may differ between species and location but the physical mechanisms of dispersal are essentially the same for all particles. Griffin et al. (2003) observed that dust particles serve as a vessel for global dispersion of bacteria and fungi. Dust borne microorganisms may play a significant role in the ecology and health of down wind ecosystems.

Rodriguez-Rajo et al. (2003) correlated annual pollen with hours of sun, temperature, wind speed and relative humidity. The results were positive with hours of sun and wind speed, and negative with rainfall and relative humidity. Abreu et al. (2003) found that distribution of pollen is very irregular throughout the year in Porto in Portugal. Al-Subai (2002) while worlcing on airborne fungi in Doha, Qatar did not find any correlation between wind direction and colony counts of fungi. Bernard (2003) opined that airborne pollen is confirmed to be a sensitive indicator of climate change. Trees appear to react stronger to climate change than grass and weeds. Jones and Harrison (2004) studied the effects of meteorological factors on atmospheric bioaerosol concentrations.

Griffin (2004) initiated a joint effort between the US Geological Surveys' Global Desert Dust and NASA's Stratospheric Cosmic Dust Programmes to identify culturable microbes from an air sample collected at an altitude of 20000m. Presence of viable microorganisms in the earth's upper atmosphere may not be unconunon.

23

Chapter 3

Material and Methods

The present study was carried out for two years from June 2005 to May 2007. Two sites were selected in Panaji city, one at Miramar, at se,a level, the second at Altinho, 2.07 km. from Miramar, at a height of 58m. The first site, Miramar, is at sea level and has sand-dune vegetation and various types of trees, shrubs and herbs along the road and unoccupied areas. It also has garden vegetation. The second site Altinho has roadside seasonal as well as perennial vegetation, besides garden plants. The third study site, ICAR (Indian Council of Agricultural Research) Complex, Old Goa, lies at a distance of 11.4 km from Miramar. It has plantation of horticultural importance as well as wild plants. Besides the above main sites, five more sites were also selected to study the seasonal variations of airspora, just for the purpose of comparative study.

They were (1) Mollem Sanctuary, a protected wildlife reserve covering 240 sq. km . of tropical evergreen forest, serni-evergreen and moist deciduous forest, situated about 46 kilometres from Panaji, (2) Cotigao Sanctuary, the southern-most wildlife reserve in Goa comprising mostly of moist-deciduous and evergreen forests (3) Mandrem, a costal village located 21 kms north of Panaji thickly populated with plantations of local commercial crops such as beetlenut and cashews along the coastline (4) Farmagudi, a pleateau located 21 kms from Panaji, comprising of mountainous terrain and (5) Margao, the commercial capital of Goa located 34 kms from Panaji to the south, mostly comprising of reseidential and commercial buildings.

The geographical location of the sites where the study was carried out, are shown in the map of Goa in Fig. 1.

24

Fig. 1 - Geographical location of the sites where the study was carried out The numbers 1 to 8 in the Fig. 1 above, indicate the following sites:

1. Miramar (N 15 °28' E 73°48') 2. Altinho (N15 °29' E 73° 49')

3. ICAR,Old Goa (N 15 °29' E 73° 55')

4. Mollem Wild Life Sanctuary (N 15 °22' E 74° 13') 5. Cotigao Wild Life Sanctuary (N 14 °58' E 74°08') 6. Mandrem (N 15 °39' E 73°44')

7. Farmagudi (N 15 °24' E 73°59') 8. Margao (N 15 ° 16' E 73 °59')

25

Air sampling was done by exposing glycerine-jelly coated slides, for 24 hours. Identification of pollen grains was done with the help of literature and reference slides prepared by collecting the flowers from the surrounding locality. Fungal spores were identified with the help of works of S.N. Agashe, P.K.K. Nair, K.R. Shivanna, T.S. Nair and others.

Composition of glycerine jelly is as follows:

1. Gelatin 50 g.

2. Glycerine 150 ml.

3. Distilled water 175 ml.

4. Phenol crystals 7 g.

Preparation of glycerine^-jelly for the gravity^-slide technique:

Gelatin and distilled water were mixed together in a beaker and boiled in a water bath till the gelatin dissolved. After half an hour, glycerine was added to the mixture and again heated for one and half hour. Phenol crystals were added and mixed thoroughly. After cooling glycerine jelly was preserved at room temperature.

Preparation of Slides:

The microslide (75x25mm) was washed thoroughly with water and soap and then cleaned with distilled water.

A major portion of the slide was coated with a thin film of glycerine jelly and a part was left uncoated for fixing a label. For coating the slide, a small lump of glycerine jelly was kept on the slide, heated on a spirit lamp flame as shown in Fig. 2.

26

Then a thin smear was made with the help of another slide as shown in Fig. 3. The slides were kept for exposure at around 9:30 a.m. in the morning and removed at 9:30 a.m. on the following day. At Miramar and Altinho the slides were exposed at a height of 12 meters, at ICAR complex it was done at a height of 8 meters and the other five sites the slides were exposed at a height of 2 meters. After exposing the coated slide for 24 hours, it was carried to the laboratory in a slide box .A drop of fresh molten glycerine was added again and a coverslip was placed. The coverslip was sealed with molten paraffin wax. The slides were later observed under the microscope as shown in Fig. 4 for identifying the pollen grains, fungal spores and other material. A total of eight sites were selected for the study. The construction of the spore trap and slide fixture are as shown in Fig. 5 and Fig. 6 respectively.

27

Fig. 2 - A small lump of glycerine jelly was kept on the slide, heated on a spirit lamp flame.

Fig. 3 - The slide was coated with a thin film of glycerine jelly.

28

Fig. 4 - Observation of exposed slides under the microscope for identifying pollen and spores.

Fig. 5 — Spore-trap showing the mounting of slide

29

Fig. 6 — Slide fixture on the spore-trap

The details of sampling frequency are presented in the Table 1.

Goa has three distinct seasons: pre-monsoon (February, March, April and May), monsoon (June, July, August and September) and post-monsoon (October, November, December and January).

This classification based on rains is a generalized one and the length of the season may vary marginally, depending on the onset, intensity and duration of monsoon.

30

Table 1- Location of study sites and sampling frequencies.

Sr. No. Name and number of Site Location Sampling Frequency

1 Miramar-I N 15°28' E 73 48'

38.30"

Everyday for two years 2 Altinho-II N15° 29' 32.37" E Everyday for two years

73° 49' 39.96".

3 Indian Council of Agricultural N 15° 29' 56.50" E Once in a month for two years Research (ICAR)-III 73° 55' 0.900".

4 Mollem-IV N 15° 22' 36.09" E Everyday for a week in each 74° 13' 41.65". season for two years

5 Mandrem-V N 15° 39' 51.25 E Everyday for a week in each

73° 44' 20.00". season for two years

6 Margao-VI N 15° 16' 44.88" E Everyday for a week in each 73° 59' 06.54". season for two years

7 Farmagudi-VII N 15° 24' 44.93"E Everyday for a week in each 73° 59' 20.77". season for two years

8 Cotigao-VIII N 14° 58' 33.054" E Everyday for a week in each 74° 08' 29.41" season for two years

Data from triplicate sampling is averaged and presented. Weather parameters were obtained from India Meteorology Department Altinho, Panaji. Data of rainfall represents the total rainfall for the entire month whereas for parameters such as temperature, humidity and wind velocity is the average for the month. These parameters are applicable to first two sites only viz. Altinho and Miramar.

31

Statistical Analysis:

Based on the aerobiological studies carried out for the two-year period between 2005 and 2007, the recorded data was subjected to statistical analysis of pollen grains and fungal spores. The data was analyzed for diversity index, evenness and species richness. This is an attempt to quantify the diversity of the airspora. All statistical calculations were based on the following formulae:

Species diversity (H')

Species diversity or diversity index was calculated using the Shannon-Weiner Index (Pielou, 1975).

H' =

E

where pi is the proportion of individuals of the ^ith species and S is the number of species.

Evenness (J')

Evenness was computed as:

J' = H' .

loge S

where H' is the Shannon-Weiner index and S is the number of species.

Species Richness (SR)

Species Richness was calculated as follows:

32

S. R. = S - 1 . loge (N)

where S is the number of species and N is the number of individuals in the collection.

Correlation

The correlation coefficient provides the magnitude of variation between two variables. It is symbolized by `r'. The correlation between various environmental parameters such rainfall, hutnidity, temperature and wind velocity and airspora was calculated by using standard Pearson's coefficient of correlation formula for twelve months data of Miramar and Altinho only. Students --`t' test was performed for each value of correlation to determine their statistical significance. The formula used for determining students-1' test was as follows:

t = r x [(n - 2) / (1 —

where r = Pearson's coefficient

n = 12 for 12-month data based on which r has been calculated

The parameters which are significantly correlated to the pollen and spore counts are determined from the standard table available for the value oft' (for n= 12), For those paramtters where the value of T is less than the threshold (1.782 for P < 0.05), the correlation is assumed to be insignificant.

33

i

Chapter 4

Results

The results obtained in this aerobiological investigation carried out in Goa during the years, June 2005-May 2006 and June 2006-May 2007, are given in this chapter.

Floristic details of plant pollen and fungal spores encountered during the study:

Pollen:

In all, the following 10 different types of pollen were encountered:

1. Acacia auriculiformis 2. Amaranthus spinosus 3. Anacardium occidentale 4. Bauhinia

sp.

5. Bouganvillea spectabilis 6. Hibiscus

sp.

7. Mangifera indica

8. Peltophorum pterocarpum

9. Poaceae

10. Tridax procumbens

Fungal Spores:

In all, the following 20 different fungi were encountered:

1. Alternaria alternata 2. Aspergillus flavus

3. Bahusakala olivaceonigra 4. Bispora

sp.

5. Cercospora elaeidis 6. Cladosporium sp.

7. Curvularia lunata 8. Didymella

sp.

9. Endophragmiella sp.

10. Fusarium sp.

11. Helminthosporium oryzae 12. Mucor sp.

13. Penicillium sp.

14. Phytophthora sp.

15. Pithomyces chartarum 16. Pleospora infectoria 17. Psammina stipitata 18. Stachybotrys sansevieriae 19. Tetraploa aristata 20. Trichobotrys sp.

Aerobiological Studies:

Site - I: Miramar

In the study site Miramar located at sea level, in the first year of study period (2005-06), 9 different types of pollen grains and 20 different types of fungal spores were identified whereas in the second year (2006-07), the types of pollen grains and fungal spores recorded were 7 and

15 respectively.

Pollen:

A total of 370360 pollen grains were trapped in the first year and 10143 pollen grains in the second year of sampling. The pollen of Poaceae dominated the assemblage of both the years of sampling and this amounted to 98.97% and 97.38% in the first and second years respectively.

Other significant pollen contributors were the genera such as Peltophorum pterocarpum, 35

Amaranthus spinosus, Acacia auriculiformis and Tridax procumbens, in both the years of sampling.

During the first year, pre-monsoon season recorded the highest number of pollen grains (218576), followed by post-monsoon (111841) and monsoon (39943) periods. The second year showed maximum number of pollen deposits in monsoon (90062), followed by pre-monsoon (5727) and post-monsoon (5664). The seasonal density and diversity are summarized in Table 2 and Table 3.

Monsoon:

During monsoon, in the first year of study, the pollen of Poaceae contributed to 99.32%

of the total catch, followed by Peltophorum pterocarpum (0.38%), Acacia auriculiformis (0.16%), Tamarindus indicus (0.09%) and Amaranthus spinosus (0.05%). In this season, maximum pollen grains were observed in the month of June. The data for the second year also showed the dominance of Poaceae pollen accounting to 98.58%, followed by Peltophorum pterocarpum (0.88%), Amaranthus spinosus (0.27%), Acacia auriculiformis (0.19%) and Tridax procumbens (0.07%). In this season, the highest number of pollen grains was observed in the month of August.

Post-monsoon:

In the post-monsoon season, the bulk of pollen grains was observed in the month of January in the first year, followed by December, November and October. Pollen grains of Poaceae were highest in contribution (97.73%) followed by Peltophorum pterocarpum (1.26%), Amaranthus spinosus (0.54%) and Acacia auriculiformis (0.08%) during the first year. In the

36

second year also, the month of January recorded the maximum number of pollen grains. Pollen grains of Poaceae (85.05%) continued to dominate the airspora, followed by Peltophorum pterocarpum (7.19%), Amaranthus spinosus (3.20%) and Tridax procumbens (2.42%).

Pre-monsoon:

During pre-monsoon, in the first year of study (2005-06), the maximum number of pollen grains was observed in the month of May. Poaceae continued to dominate the assemblage, amounting 99.55%, followed by Peltophorum pterocarpum (0.33%), Amaranthus spinosus (0.09%) and Tridax procumbens (0.02%). The study of airspora in the second year (2006-07) again showed the dominance of Poaceae, followed by Peltophorum pterocarpum and Amaranthus spinosus. Maximum number of pollen was in the month of February, followed by April, March and May.

Fungal Spores:

The study of aerospora during the first year (2005-06) exhibited a total number of 157451 fungal spores trapped, belonging to 20 species. The data in the second year (2006-07) revealed a catch of 259910 fungal spores belonging to 15 species. The results obtained are shown in Table 4 and Table 5. Monsoon recorded the maximum number of spores, followed by post-monsoon and pre-monsoon. ^Bispora sp. contributed to 52.45% of the annual total, followed by Cercospora elaeidis (41.12%), Aspergillus flavus (2.59%) and Mucor sp. (1.51%) in the first year. During the second year of study Bispora sp. continued to dominate the assemblage with 98.46%, followed by Aspergillus flavus (0.64%), Cercospora elaeidis (0.27%) and Fusarium sp. (0.21%). In both

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the years, the maximum number of spores was caught in the monsoon season, followed by post- monsoon and pre-monsoon.

Monsoon:

During monsoon, in the first year, the month of July recorded the highest catch, followed by June, September and August, the total fungal spores counted was 151516. The assemblage was dominated by the spores of

sp. (53.08%),

(42.2%),

sp.

(1.57%) and

sp. (0.34%). In the second year, the month of July recorded the maximum spores, followed by August, June and September. The total number of spores deposited was 257094. The assemblage was again dominated by

sp. (92.51%), followed by

(0.59%),

sp. (0.10%) and

(0.08%).

Post-monsoon:

In the post-monsoon season, a total of 4247 spores were found deposited on the slides during the study in the first year. The month of October recorded the highest number of spores, followed by November, December and January.

sp. continued to dominate the assemblage followed by

sp.,

sp. and

sp. The second year of study revealed a total catch of 2479 fungal spores from the air. The highest number of spores was recorded in October. The conidia of

sp. were maximum in the assemblage, followed by

and

sp.

Pre-monsoon:

The pre-monsoon of first year of study showed 1688 fungal spores. The spores of

sp. were highest in content (55.09%) followed by

(14.45%),

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