International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 2(1), 12-18, January (2013) Int. Res. J. Environment Sci. International Science Congress Association 12 Incidences of Arbuscular Mycorrhizal Fungi (AMF) in Urban Farming of Mumbai and Suburbs, India Kelkar Tushar S. and Bhalerao Satish A. Environmental Sciences Research Laboratory, Department of Botany, Wilson College, Mumbai, INDIA Available online at: www.isca.in Received 27th November 2012, revised 25 December 2012, accepted 10 Januray 2012Abstract Mumbai, economical capital of India is its true sense is Mayanagari, where the major problem is of space for accommodation and so as land under cultivation is rare in occurrence. The Local train is a life-line of Mumbai. It passes from the major part of Mumbai city and its suburbs. Near the tracks some area of land might be available for urban agriculture. To support day today needs of fresh vegetables, majority of the time such land near railway tracks are utilized. It is the waste land near tracks utilized for cultivation of leafy vegetables like fenugreek (Trigonella longiceps), chavali (Vigna unguiculata), fruit vegetables like bhindi (Abulmoschus esculantus), and some Chenopodiaceae members. Such soils are often of very poor quality for cultivation practices. Irrigation is often done from gutters and waste water from washing and garage water. In such disturbed habitats incidences of mycorrhizal fungi are more in terms of percent colonization. In present investigation, roots of such commonly cultivated crops like bhindi, chavali, and fenugreek were collected from different locations from Central Railway side tracks and screened for % colonization by mycorrhiza fungi. Similarly, rhizospheric soil samples were also collected and detail investigations were carried out like physical parameters such as soil pH, soil moisture, and organic matter. Samples were screened for Arbuscular Mycorrhizal Fungi (AMF) spores also and spore density was calculated for each sample. Lastly, all such parameters were analyzed for any correlation present amongst them. Keywords: Disturbed habitats, urban agriculture, AM fungi, AMF spores, bhindi (Abulmoschus esculantus), chavali (Vigna unguiculata), and fenugreek (Trigonella longiceps) % root colonization, AM spore density. Introduction Mumbai, economical capital of India is its true sense is Mayanagari, where the major problem is of space for accommodation and so as land under cultivation is rare in occurrence. The Local train is a life-line of Mumbai. It passes from the major parts of Mumbai city and its suburbs. Near the tracks some area of land might be available for urban agriculture. Such areas are generally utilized by slum people for the accommodation, for garbage disposal and for as their backyards. Railway stations of Mumbai suburbs like Thane, Dombivali are quite large stations and much more such spaces are observed, whereas in Mumbai area like Masjid railway station is hardly having such space. In short more we move towards Mumbai Chattrapati Shivaji Terminus Railway platforms, the availability of space is problem whereas if we go to opposite direction of it like Parel, Bhandup, Thane, Mumbra etc the available land for urban agriculture becomes larger. To support day today needs of fresh vegetables, majority of the time such land near railway tracks are utilized. It is the waste land near tracks utilized for cultivation of leafy vegetables like fenugreek (Trigonella longiceps), chavali (Vigna unguiculata), fruit vegetables like bhindi (Abulmoschus esculantus), and some Chenopodiaceae members. Such areas are managed hardly for such cultivation. No proper irrigation system and resource is available hence in such practices any water source is utilized for irrigation purpose. Such soils are often of very poor quality for cultivation practices. Irrigation is often done from gutters and waste water from washing and garage water. The word mycorrhiza means fungal root derived from two Greek words as mycos meaning fungus and rhiza meaning root. Mycorrhiza can be defined as a symbiotic association for one or both partners between a fungus and a root of living plant that is primarily responsible for nutrient transfer. The mycorrhizal fungi proliferate in both roots and soil. The soil borne extra radial hyphae take up the nutrition from the soil and transport them to the root system of the plant. It helps in increasing the surface area of absorption of the plant root system. In the soil with low moisture and low nutrient contain, this process leads to improve the plant growth and reproduction. The plant showing mycorrhizal association has more ability to tolerate the environmental stress than the non mycorrhizal plants. Exceptionally wide range of plants in different ecosystems shows association with AM fungi. The latter play a major role in better nutrition, species diversity and survival. Amongst the Angiosperms, about 90 per cent of the families develop AM association. The occurrence of AM fungi differs qualitatively International Research Journal of Environment Sciences______________________________________________ISSN 2319–1414 Vol. 2(1), 12-18, January (2013) Int. Res. J. Environment Sci. International Science Congress Association 13 and quantitatively with the change in edaphic factors and type of vegetation. Ecological factors which influence the colonization of AM fungi in the soil are soil pH, temperature, moisture, organic matter content in the soil and soil pollutants. Changes in the soil pH greatly affect the development and functioning of AM fungi. Different species and strains of AM fungi show different responses to soil pH. Higher fungal spore density in acidic soil, while in alkaline soil the spore density is low has been reported. Higher temperature results into high root colonization in temperate zone whereas it can be just a reverse in tropical region. AM fungi prefer optimum soil moisture for sporulation and growth. Because of it, during winter and summer spore density is low as comparing to rainy season in which it is quite high. Organic matter and soil fertility also play an important role in growth and sporulation of AM fungi. In low fertile soil, AM fungi spore density is higher to that of high fertile soil where it is quite low. Changes in soil fertility due to amendments with mineral fertilizers or organic matter markedly affect the activity and survival of AM fungi. They can grow saprophytically around decaying root fragments and other organic matter. Nitrogen can stimulate or suppress root colonization by AM fungi and sporulation by changing the soil pH. In the presence of sufficient amount of Nitrogen in the soil, addition of Phosphorus suppresses root colonization. More amount of AM colonization is observed in P deficient soil. In present investigation, roots of such commonly cultivated crops like bhindi chavali, and fenugreek were collected from different locations from Central Railway side tracks and screened for % colonization by mycorrhiza fungi. Similarly, rhizospheric soil samples were also collected and detail investigations were carried out like physical parameters such as soil pH, soil moisture, and organic matter. Samples were screened for Arbuscular Mycorrhizal Fungi (AMF) spores also and spore density was calculated for each sample. Material and Methods For the present investigation sample collection was the main fundamental aspect. Since in Mumbai, central railway has sprayed like a network and almost 70 per cent of the stations are having such lands under urban agricultural practices, random sampling was done. The plants with root system along with rhizospheric soil were carefully collected in clean, unused plastic bags of suitable size. The samples are carefully labeled showing the records like date of collection, time, location, name of the host plant etc and were brought in the laboratory. After bringing the samples in laboratory, roots were carefully separated from entire plants, tapped gently to separate soil particles which were adhered to it (which was put in respective soil samples). The root samples were washed under tap water to clean and stored in 70 per cent of alcohol until further use. Soil samples were dried under sunlight and stored in well labeled, sterilized plastic bags. Likewise the samples were collected from different locations with the gap of almost only half a day as all the sites were situated on central railway side areas and there is a practical threat of harvest for day to day consumption and for selling to local distributors by plant growers. Percentage root colonization was calculated after staining procedure7,8. The roots of collected plants were washed thoroughly with running tap water for removal of alcohol which was used as preservative. The properly cleaned roots were subjected to staining procedure. Roots were subjected to 10 per cent of potassium hydroxide and heated in water bath at 90 C for one hour to clear the tissue. Such cleared roots were washed with distilled water several times to remove the traces of alkali. In case of dark pigmentation of roots, they were treated with three per cent hydrogen peroxide for bleaching. It took 5 to 30 minutes depends upon the degree of pigmentation. This step was eliminated in the case of roots which are white or non pigmented. Once again roots were washed thoroughly for several times with distilled water. Washed roots were kept in 1 per cent hydrochloric acid for about 18 hours to neutralize the effect of alkali used for cleaning the root tissue. Acidification was followed by washing several time the treated roots to remove traces of acid. The roots were then kept in acid glycerol containing 0.05 per cent aniline blue in test tune. Then it was autoclaved for 15 minutes under the pressure of 15 labs at 121 C. After that, excessive aniline blue was drained off and excessive stain was removed from stained roots by using acid glycerol. The stained roots were stored in amber coloured bottle in acid glycerol until further use to avoid destaining. The roots were cut into small pieces approximately 1 cm in length and mounted on micro slides using glycerin as mounting medium. After placing the cover slips, the slides were observed under low power of objective of 10 X magnification to confirm the presence of mycorrhizal hyphae and high power objective of 40 X magnification to observe the presence of vesicles, spores etc. the slides were sealed with nail polish. The per cent root colonization was calculated by following formula 9.  \n \n\rNo. of root pieces showing infection \n  Isolation and quantification of AM spores was carried out by wet sieving and decanting method10. 25 g of sundried rhizospheric soil of different soil of different plants collected at different localities was taken in separate beakers. Half a litre of water and a pinch of soap powder was added to this. The solution was stirred and was allowed to stand for half an hour. The soil solution was then filtered through sieves of 500, 250, 150, 105 and 55 mm mesh which were kept one above the other. The spores and soil particles which settled on the surface of 150, 105 and 55 mm mesh sieve were washed and collected in separate beakers. The water was again filtered through whatman filter no. 1. This paper was observed under stereoscopic binocular microscope to count the number of spores. Different spores were examined for their taxonomic status by using standard key11. International Research Journal of Environment Sciences______________________________________________ISSN 2319–1414 Vol. 2(1), 12-18, January (2013) Int. Res. J. Environment Sci. International Science Congress Association 14 Rhizospheric soil sample were analyzed for its various physico – chemical properties like soil pH, organic matter content were estimated. Soil pH was determined by using pH meter model number EQ 614. Organic matter content was estimated12. Observations: The plants studied for AM fungi colonization are listed below with the locations from where they were collected table 1. Plants were studied for AM fungi association in terms of per cent root colonization figure 3 and no. of AM spores per 25 g of rhizospheric soil figure 4. The observations are tabulated in table-2 similarly, rhizospheric soil samples were analyzed for physico – chemical properties such as pH of soil, organic matter content and shown in table-3. Table-1 Locations and plants collected for sampling Location / Site Name of Plants collected Thane Trigonella longiceps Vigna unguiculata Mulund Trigonella longiceps Vigna unguiculata Abulmoschus esculantus Spinacia oleracea Bhandup Trigonella longiceps Vigna unguiculata Abulmoschus esculantus Spinacia oleracea Kanjur marg Spinacia oleracea Parel Trigonella longiceps Vigna unguiculata Abulmoschus esculantus Spinacia oleracea Kari road Trigonella longiceps Vigna unguiculata Abulmoschus esculantus Spinacia oleracea Table-2 Percent root colonization and no. of AM fungal spores Location / Site Name of Plants collected % Root colonization No. of AM spore / 25 g sundried rhizospheric soil Thane Trigonella longiceps 85.2 116 Vigna unguiculata 90.0 68 Mulund Trigonella longiceps 80.0 108 Vigna unguiculata 89.12 78 Abulmoschus esculantus 68.0 65 Spinacia oleracea 0 35 Bhandup Trigonella longiceps 84.0 99 Vigna unguiculata 88.0 67 Abulmoschus esculantus 69.2 66 Spinacia oleracea 0 31 Kanjur marg Spinacia oleracea 0 39 Parel Trigonella longiceps 88.7 100 Vigna unguiculata 90.2 78 Abulmoschus esculantus 65.9 78 Spinacia oleracea 0 24 Kari road Trigonella longiceps 87.9 109 Vigna unguiculata 82.0 78 Abulmoschus esculantus 67.8 67 Spinacia oleracea 0 29 International Research Journal of Environment Sciences______________________________________________ISSN 2319–1414 Vol. 2(1), 12-18, January (2013) Int. Res. J. Environment Sci. International Science Congress Association 15 Table-3 Physico – chemical properties of rhizospheric soil samples Location / Site Soil pH Organic content of soil (%) Thane 6.2 2.33 Mulund 6.0 2.1 Bhandup 5.7 1.9 Kanjur marg 6.0 2.2 Parel 6.9 3.0 Kari road 6.0 2.77 Figure-1 Map of study area International Research Journal of Environment Sciences______________________________________________ISSN 2319–1414 Vol. 2(1), 12-18, January (2013) Int. Res. J. Environment Sci. International Science Congress Association 16 (http://www.mumbainet.com/images/cityinfo/railmap.gif) Figure-2 Location map of Mumbai (shows location in map from where samples were collected) Figure-3 Per cent root colonization of samples according to the locations  \n\n    \r\r \r \r\n  \r\n      International Research Journal of Environment Sciences______________________________________________ISSN 2319–1414 Vol. 2(1), 12-18, January (2013) Int. Res. J. Environment Sci. International Science Congress Association 17 Figure-4 No. of AM fungal spores per 25 g of rhizospheric soil sample location wise Results and Discussion All the rhizospheric soil samples collected were showing almost 90 % of the moisture content, hence the detail comparison is not carried out. This must be because of enough amount of irrigation was done in every locations though that water was not of safe and good quality. All soil samples were acidic in pH, whereas all of them show very poor quality in terms of organic matter content. It can be one of the main reasons to have a good quantity of AM fungal spores which ranges from 24 as least in sample of Kanjur marg where as maximum in Thane sample showing 116 AM fungal spores per 25 g sundried rhizospheric soil samples. Very interestingly in all the samples of Kanjur marg which were collected from rhizospheric soils of Spinacia oleracea, presence of AM fungal spores though the spore count was very low. This might be because of absence of suitable host. It has been reported by many workers as Chenopodiaceae members don’t show AM fungi colonization. In the present investigation it was found that all the samples of Trigonella longiceps shows highest root colonization as well as highest spore number in rhizospheric soil, followed by Vigna unguiculata, Abulmoschus esculantus. This variation in observations from site to site indicates the variation because of physico – chemical parameters which differ slightly. But with respect to species observations are more or less same. No fixed correlation could be drawn since many factors like uneven irrigation, duration of irrigation, unexpected harvest for local markets are operative at same time. Glomus mosseae is the most common in occurrence in all soil samples with highest spore density, where as Gigaspora sp and Acaulospora sp were also observed in all soil samples. There occurrence were in comparison less than Glomus mosseae in terms of spore density. Conclusion Based on the above observations and results conclusion can be drawn that such unused lands which are on sides of railway tracks can be potentially utilized for urban agriculture. Mycorrhizae are proved to be good enough to sustain in very poor fertile soil. So for such growers, ideas are to be provided to use mycorrhizal inoculums for their cultivations. Such commercially produced inoculums are easily available in market. Irrigation system should be developed properly which will supply fresh, pollutant free water for plants.AcknowledgementAuthors are thankful to DR. V. J. Sirwaiya, Principal, Wilson College, Mumbai for providing lab facilities. They are also grateful to teaching and non teaching staff of Department of Botany for their constant support. References 1.Brundrett M.C. Diversity and classification of mycorrhizal associations Biological Review 79, 473–496 (2004)2.Sylvia D.M. and S.E. Williams. Vesicular-arbuscular mycorrhizae and environmental stress, In Linderman, R.G., and G.J. Bethlenfalvay (Eds.). Mycorrhizae in Sustainable Agriculture. Special publication No. 54, American Society of Agronomy, Madison, WI. 101–124(1992)3.Smith F.A. and S.E. Smith Structural diversity in (vesicular)—arbuscular mycorrhizal symbioses New Phtol137, 373 – 388 (1996) \r    \r\n\r\n\n\n\r\r\r\n\r\n    \r       ! International Research Journal of Environment Sciences______________________________________________ISSN 2319–1414 Vol. 2(1), 12-18, January (2013) Int. Res. J. Environment Sci. International Science Congress Association 18 4.Dalal S. and Hippalgaonkar K.V. The occurrence of vesicular arbuscular mycorrhizal fungi in aerable soil of Kokan and Solapur Mycorrhizae: Biofertilizers for the Future (eds.) Adholeya A. Singh S. Tata Energy Reseach Institute, New Delhi, India, 3-7 (1995)5.Hepper C.M., Warner NA Organic matter in the growth of vesicular-arbuscular mycorrhizal fungi in soil, Trans Br Mycol Soc,81, 155–156 (1983) 6.Sylvia D.M., and L.H. Neal Nitrogen affect the Phosphorus response of VA mycorrhiza, New Phytologist, 115(2), 303–310 (1990) 7.Koske R.E. A modified procedure for staining roots to detect VAM fungi, Mycol. Res, 92(4), 486-48l (1989)8.Carol Grace and D.P. Stribley A safer procedure for routine staining of vesicular – arbuscular mycorrhizal fungi, Mycol. Research, 95(10), 1160-1162 (1991)9.Nicolson Nicolson’s formula Mycorrhiza News12(2)(1955) 10.Gerdeman J.W. and T.H. Nicolson Spores of mycorrhizal Endogene sp. extracted from soil by Wet-sieving and decanting, Trans. Br. Mycol. Soc.,46(2), 235-244 (1963)11.Schenk, N.C. and V. Perez Manual for the identification of VA-Mycorrhizal fungi, 3rd Edition (1989)12.Blackman and Walky An examination of degjjareft methods for determining soil organic matter and a proposed modification of the chromic acid titration method, Soil Sci., 37, 29–38 (1935)