JOURNAL OF THE BOTANICAL SOCIETY, UNIVERSITY OF SAUGOR (Formerly Bulletin of the Botanical Society)

Volume 44, 2014

Published By The Botanical Society, University of Saugor-470003, India Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

BOTANICAL SOCIETY DEPARTMENT OF BOTANY Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.)

OFFFICE BEARERS (2012-1013)

Patron : Prof. D.C. Atri President : Prof. P. Mehta Vice President : Prof. A.K. Kandya Treasurer : Dr. N.P. Bhalla General Secretary : Dr. S.K. Yadav Research Scholar Ramnaresh Chanpuriya Omkar Salanke

Academic Secretaries: Prof. (Mrs.) J. Dubey Dr. A.S. Mishra Dr. N.P. Bhalla

Executive Editor : Prof. A.N. Rai Dr. Deepak Vyas

Joint Editor : Dr. (Mrs.) A. Mehta

Business Manager : Prof. A.K. Kandya Dr. N.P. Bhalla

Editorial Board : Prof. P.K. Khare Dr. Poonal Deharia Dr. A.J. Biswas

Librarian : Dr. N.P. Bhalla Dr. A.J. Biswas Secretary : Praveen Pandey (M.Sc. III)

Joint Secretary : Archna Kushwaha (M.Sc. II)

Excursion : Nishant Singh (M.Sc. I)

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 2 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Efficiency of different casing mixtures and their thickness on growth and production of Calocybe indica Anjuli Chaubey* Poonam Dehariya and Deepak Vyas Lab of Microbial Technology and Plant Pathology, Department of Botany, Dr. H. S. Gour University, Sagar (M.P.) e-mail: [email protected]

ABSTRACT Seven casing materials viz., Farm yard manure (FYM), Spent compost (SC), Garden soil (GS), Sandy soil (SS) alone and in combination as FYM+SC (1:1), FYM+GS (1:1) Sandy Soil (SS) and FYM+SS (1:1) were analysed with different treatments (Formaline (4%), autoclaved (15 ibs) and untreated (control) to see the effect on growth and production of C. indica. Effect of different casing thickness was also investigated. 2.5 cm thickness was found optimum to obtained higher yield of Calocybe indica (401.33 g/kg dry substrate). 4% formaline treated mixture yielded better in comparison to other combinations. Interestingly, when FYM was treated with 4% formaline gives better growth and higher yield of C. indica.

Key words: C.indica, casing mixtures , casing thickness, treatment INTRODUCTION White milky mushroom (Calocybe indica), natïve to India, was first described by Purkayastha and Chandra (1974). The ability of this mushroom to grow at temperature of about 30-350C, accompanied by its excellent shelf –life makes it highly attractive market venture among the growers. Milky mushroom cultivation involves a number of operations. Once the spawn run is complete, the crop enters into reproductive phase leading to production of fruiting bodies. Even after the colonization of substrate, fructification will not take place unless the colonized substrate is covered with casing layer. The process of applying casing layer over the compost bed is called Casing (Pandey, 2008). Casing is an agronomic practice to cover the top of mushroom beds after spawn run with layer of appropriate soil mixture (Tandon et al., 2006). In our country different types of casing materials are used in accordance with the local availability and suitability. The composition of casing mixture determine its quality (texture, structure, pH, water holding capacity, (C:N) ratio, etc.), which directly affect the mycelial growth in casing layer and initiation of fruiting bodies (Tewari, 2005). Adjustment in thickness of casing layer accordance to the quality of casing mixture is necessary. The thickness of casing layer not only affects initiation of fruiting bodies and their development but also the duration of crop and fruiting body yield (Shukla, 2007).

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 3 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

MATERIALS AND METHODS The culture of Calocybe indica was procured from the Mushroom Research and Training Centre, Pantnagar. The culture was maintained on MEA and incubated at 28+ 20C temperature. Cultivation was carried out in polythene bags of 60×30cm size and 100 gauge thickness. Spawning is done @ 4% wet weight basis of substrate employing thorough method of spawning (Shukla, 2008). The bags were kept in the crop room. For spawn run relative humidity and temperature were maintained between 85-90% and 32-350C, respectively. The completely colonized substrate was cased with different casing mixtures. Seven casing materials were collected namely Farm yard manure (FYM), Spent compost (SC), Garden soil (GS), Sandy soil (SS) alone and in combination as FYM+SC (1:1), FYM+GS (1:1) and FYM+SS (1:1) were treated different methods viz., with 4% formaline, autoclaved 15 lbs and untreated. The different casing thickness i.e. 1 cm,1.5 cm., 2.0 cm, 2.5 cm, 3.0 cm and 3.5 cm. were applied on the bags to see the effect on growth and production. The bags were covered with newspaper to prevent infestation by insects and weed moulds. Light watering was done twice a day to maintain 60% moisture on the bed surface. Data on days for pin head appearance, Number. of fruiting body, yield and BE were recorded. The experiment repeated three times. Statistical analysis was done by software systat 12. Results: Effect of seven casing mixtures with different treatments on yield and sporophore development of C. indica was presented in Table (1) The results show that appearance of fruiting body of test organism treated with different casing mixtures showed significant variation. As it is clearly evident FYM treated with 4% formaline solution was found better casing substance which provide conducive environment to the mushroom for early pin head appearance, and finally gives higher yield in comparison to other casing mixtures. In contrast to this SS was found poor casing mixture which responses slowly and do not provide very good atmosphere to the mushroom mycelium, resulting slower growth and poor yield of the mushroom. To obtain better yield of the mushroom not only casing material is prerequisite but more important thing is the thickness of the casing material maintained in the bags. In Table (2) a total of six casing thickness were analyzed to observe their effect on the growth and yield of Calocybe indica. The result obtained from this present experiments that thickness has played a significant role on the growth of C.indica. Different bags were maintained with different thickness and the result obtained the experiments suggest that 2.5 cm. thickness was optimum to obtained better growth and highest yield of the test organism.

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 4 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Discussion: Present findings indicate that among the casing mixtures FYM, Spent compost alone and their combination gave significantly higher mushroom yield . Among all the mixtures FYM had the highest water holding capacity (130%) and spent compost (105%) similarly maximum pore space was found in FYM followed by spent compost (Tandon et al., 2006). Thus This mixtures and their combinations have improved the physical and chemical properties of the casing media which resulted in better yield of mushroom. Effect of casing mixtures and treatments on yield of Calocybe indica was investigated by different workers and found variable results. Saini and Prasher (1992) suggested casing formulation developed from FYM + waste compost + soil (2:1:1, v/v) for better yield. While, Pandey and Tewari (1994) suggested use of garden soil, coir dust and FYM as casing material for cultivation of C. indica. Singh et. al., (2007) found that the casing prepared using spent compost, farm yard manure, sand and garden soil (1:1:1:1) gave the highest yield. Bhatt et. al., (2007) found that farm yard manure + sand (1:1) as casing mixture resulted in the highest yield of Calocybe indica. According to present study, FYM treated with formalin 4% solution was found best this is accordingly to the earlier work done by Mantel (1973), Saxena and Gupta (1986). Since casing thickness lead an important role for the growth of mycelium and higher yield of mushroom. Therefore, it was mandatory to investigate thickness of casing material. Because, thickness provide adequate moisture, aeration and protection. If thickness was less it affects adversely, due to lack of moisture, greater aeration and poor protection resulting causing poor growth of the mushroom. In contrast to this if thickness is high then it may provide greater moisture but poor aeration and conducive environment for the fungal weeds will negatively effect which also affect adversely on the growth and yield of the mushroom. Thus it is very important to achieve optimum thickness for the growth and yield of the mushrooms. Our results indicate 2.5 cm thickness is the optimum thickness which gives better environment for the growth of the mushrooms. Almost similar thickness 2.0 cm was used by Krishnamoorthy & Muthuswami(1997) to get the highest yield of C. indica.

Acknowledgement : We are thankful to Head, Department of Botany for providing laboratory facilities and A.C. thanks U.G.C. for financial assistance.

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 5 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

REFERENCES Pandey, V., (2008). Impact of physico-chemical properties and microbial dynamics of casing on sporophore development and yield of Calocybe indica P&C. Msc.Thesis , G.B.P.A. U. , Pantnagar (Uttrakhand). Tandon, Gayatri, Sharma, V.P. and Jandaik, C.L.(2006). Evaluation of different casing materials for Calocybe indica cultivation. Mush. Res. 15(1):37-35.

Tewari, R.P. (2005) . From Director’s Desk. Mush. Newsl., National research centre for mushroom, Chambaghat, Solan , pp. 8 Shukla, P.K. (2007). Effect of casing soil thickness on crop duration and yield of milky mushroom (Calocybe indica P.&C.). Indian Phytopath. 60 (4): 537-539 . Shukla, P.K. (2008). Effect of soil and manure ratio of casing soil on crop duration and yield of milky mushroom (Calocybe indica). J. of Mycol and Pl. Pathol. 38 (1): 47-50. Saini, L.C. and Prashar, R.D. (1992). Casing media in relation to the yield of button mushroom (Agaricus bisporus). Agri. Sci. Digest. 12(1):13-14. Pandey, M. and Tewari, R.P. (1994). Evaluation of casing material for Calocybe indica cultivation. Paper presented in National Symposium on Mushroom held at NCMRT, Solan, April 8-10. pp. 74. Singh, Mandvi; Singh, A.K.; Gautam, R.K. (2007). Effect of casing and supplementation on yield of milky mushroom (Calocybe indica) Indian Phytopath. 60(2) 191-193. Bhatt, P.; Kushwaha, K.P.S. and Singh, R.P. (2007). Evaluation of different substrates and casing mixtures for the production of Calocybe indica. Ind. Phytopath. 60: 128-130. Mentel, E.F.K.(1973). Casing soil made from spent compost. Ind. J. Mush. 1:15-16. Saxena, S. and Gupta, Y. (1986). Annual Report, NCMRT, Solan. pp. 29-30. Vijay, B.; Gupta, Y.; Upadhyay, R.C. and Gupta, Y. (1988). Effect of casing thickness on yield of Agaricus bisporus. Ind J. Mycol. Plant Pathol.18 (2):209-210. Krisnamoorthy, A.S. and Muthusamy, M. (1997). Yield performance of Calocybe indica (P & C) on different substrates. Mush. Res. 6 (1):29-32.

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 6 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Table 1 : Effect of casing mixtures and their treatments on yield of Calocybe indica

Casing mixture Treatment Days for No. of Yield (g/kg pinhead fruiting dry substrate) initiation body Farm Yard F 11.00 40.66 475.00 Manure A 10.00 32.00 400.66 (FYM) C 9.66 11.00 221.66 Mean 10.22 27.88 365.77 Spent Compost F 14.66 31.66 438.00 (SC) A 12.00 18.33 324.33 C 10.00 15.66 213.33 Mean 12.00 21.88 310.00 Garden Soil F 15.66 19.00 305.00 (GS) A 12.33 11.66 265.00 C 11.00 8.33 183.33 Mean 12.99 14.99 251.11 Sandy soil (SS) F 15 17.00 213.33 A 14.66 14.66 166.66

C 14.00 4.66 103.33 Mean 14.55 12.10 161.10 FYM+SC (1:1) F 13.00 34.00 451.66 A 11.00 25.66 338.33 C 9.00 13.33 220.00 Mean 11.00 24.33 336.66 FYM+GS(1:1) F 13.66 19.66 348.33 A 11.33 15.33 280.00 C 10.00 8.33 183.33 Mean 11.66 18.44 270.55 FYM+SS (1:1) F 14.00 24.00 203.33 A 12.00 18.66 201.66 C 13.00 4.66 118.33 Mean 13.00 15.77 174.44

CD(P=0.05%) Casing mixture (a) 1.08 2.50 Treatment(b) 1.00 2.45 Interaction (a×b) 2.34 5.66 F- formalin, A- autoclaved at 15 lbs, C-control (without treatment)

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 7 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Table 2: Effect of casing soil thickness in growth and yield of Calocybe indica

Casing Pinhead Total no. Yield thickness (cm) initiation fruiting body (g/ kg dry substrate) (days) 1.0 11.67 21.33 259.58 1.5 10.67 22.00 297.00 2.0 8.33 23.67 376.83 2.5 9.33 24.33 401.33 3.0 10.33 21.33 365.75 3.5 12.00 21.33 363.50 SEm (±) 0.83 2.20 4.51 CD (P=0.05%) 2.58 7.07 13.91

Fig.1: Effect of Casing mixtures and treatments on biological efficiency of C. indica

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10 Biological efficiency (%) efficiency Biological

0 F F F F F F F A C A C A C A C A C A C A C Mean Mean Mean Mean Mean Mean Mean

Farm Yard Spent Garden Soil Sandy soil FYM+SC (1:1) FYM+GS(1:1) FYM+SS (1:1) Compost (GS) (SS) (SC) Casing Mixtures

Fig.2: Effect of casing thickness on biological efficiency of C.Indica

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Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 8 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Ecological Observations of Some Forest Plant Species in Sagar District of M.P. A.J.Vishwas*, P.K.Tiwari, Y.S. Thakur, and S.P. Bajpai Department of Botany Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.)

ABSTRACT The paper deals with some ecological investigations of biodiversity, topography, geography, soil and climatic conditions of seven selected forest sites of Sagar district. The study area also shows diversity in natural regeneration of plants. Amongst various species the sources of natural regeneration were found to be seedlings, saplings, coppices, root suckers. Continuous type of regeneration seems to be the characteristic of biologically disturbed forests however stagnant type seems to be the characteristic in relatively less disturbed forests.

INTRODUCTION Sagar District is suitated in the centre of Madhya Pradesh. It is situated between 23o.9' and 24o.27' Northern latitude and 79o.3' and 79o.57', Eastern latitudes. It covers 1.7% area of Madhya Pradesh and has an area of 7322 Sq.Km.. The north to south stretch of Sagar district is approximately 144.8 Km. and width is 80.46 Km. The total area of Sagar District is about 7322 Sq.Km., one third area of which is covered by forests. Forests of Sagar are very heterogeneous in their composition, quality, density and extent. Prevalent variation pose difficulty in precisely delimiting the distribution of forest stands into well recognized forest types of exactly alike and homogeneous & composition for the purpose of classification and description of the types. Even the crop standing in the same localities are not homogeneous in their quality, density and composition. Microclimatic differences produced due to variation in slope aspect and proximity to water sources, cause a very perceptible biodiversity in the vegetation. Thus, overall an area with fairly uniform climatic, microclimatic conditions affect upon the vegetation resulting in segregation of species from distinct stands. Main factors governing the distribution of forest types and stand results soil physiography of the tract and biotic influences.

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 9 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

MATERIALS AND METHODS Field investigations were conducted during June 2001 to May 2004 and seasonal visit for recording first hand information regarding the economic value of climatic conditions, topography, geography & soil of plants, in the area was done. The collected information was cross checked with the help of available literature. Biodiversity and similarity amongst all seven sites regarding species composition, topographic situation, climatic conditions and soil was obtained by using "community coefficient" formula (Kulczynski, 1937).

RESULTS AND DISCUSSION The topography of Sagar district is of very undulating type with low rising hills scattered all around. Sagar district is located at the southeastern part of huge Vindhyan plateau. Basalt and Vindhyan sand stones are the two main rocks in Sagar district. Talus are generally formed by the Vindhyan rock fragment, which detach out the crops due to the gravitational force, wind, water, physical weathering and roots of tree, and are carried down through slopes. According to Wadia (1948), basalt is the igneous formation of late cretaceous, while Vindhyan sandstone is a sedimentary formation of Proterozoic period. Besalt are more susceptible to physical as well as chemical weathering in comparison to Vindhyan sandstone. The consequence of the characteristic spheroidal weathering leads to the production of round boulders of different sizes. Accumulation of boulders can be observed particularly on steep slopes due to the removal of top soil on account of destruction of forest vegetation, overgrazing and subsequent erosion of soil in the area covered with thick vegetation. The soil remains accumulated upto a depth of few cm. to half a meter in area where soil erosion is not conspicuous. The topography of the sand and the vegetation cover are the two main factors determining the thickness, colour, texture and chemical properties of soil (Tiwari, 1994). At the foot hills the soil layer is black in colour, which remains waterlogged through out the rainy season, as it is non-porous. Such a type of soil is fertile and varies in thickness. At the slope of plateau, the soil is light brown to reddish brown due to laterization. Soil derived from the Basalt hills are slightly alkaline with more of organic matter, phosphorus, potassium and calcium, however soils derived from sand stones are slightly acidic with more nitrogen (Rathore, 1968).

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 10 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

CLIMATIC CONDITIONS Climate of Sagar is seasonal with three well-marked seasons viz., rainy, winter and summer on the basis of temperature, rainfall and relative humidity. Rainy season According to climatic conditions, rainy season in Sagar begins from the middle of June and continues up to September. It is strictly confined to four months. The characteristics of this season are heavy rainfall, high relative humidity, high temperature and least diurnal variations. Winter season Period of winter starts from the middle of October and ends in February. It is characterised by low temperature and moderate relative humidity. Occasional showers are received in the month of December and January, which cause a further fall in the day temperature. Summer season: The period starting from middle of February till mid June and is characterized by high temperature and low humidity. The average rainfall of Sagar during June 2001to 2004 is given in table 1 fig. 1.

Table-1: AVERAGE MONTHLY RAIN FALL (mm) AT SAGAR DURING STUDY PERIOD

Months Years 2001 2002 2003 2004 January - 0.00 0.00 36.00 February - 24.75 87.75 0.00 March - 0.00 2.25 0.00 April - 0.00 0.00 0.00 May - 0.00 0.00 0.00 June 225.00 45.00 92.25 0.00 July 375.25 31.50 294.75 - August 195.75 479.25 335.25 - September 45.00 130.50 438.75 - October 56.25 56.25 6.75 - November 2.25 11.25 0.00 - December 0.00 2.25 0.00 - Total 899.50 780.75 1257.00 36.00

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 11 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Fig. No. 21 Average monthly rainfall in (mm) at DamohSagar during study period

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0 July May April June March August January October February November December September

2001 2002 2003 2004

On the basis of past three years temperature records, it is moderate with average minimum and maximum temperature of 10.8oC and 42.1oC. In summer, temperature goes up to 47.63oC and in winter it comes down to 10.35oC. Monthly mean max. and min. temp from June 2001 to May 2004 is given in table 2 fig. 2.

Table-2: AVERAGE MONTHLY TEMPERATURE (oC) AT SAGAR DURING STUDY PERIOD

Months Year Average 2001 2002 2003 2004 Min. Max. Min. Max. Min. Max. Min. Max. Min. Max. January - - 11.48 25.72 10.59 25.81 10.35 25.65 10.80 25.72 February - - 12.67 26.73 14.31 29.20 11.91 26.80 12.96 25.57 March - - 18.94 34.38 16.84 31.36 19.06 35.23 18.28 33.65 April - - 23.59 37.87 23.37 37.01 21.18 35.95 22.28 36.94 May - - 25.45 40.26 26.45 42.37 26.64 41.57 25.51 41.40 June 26.45 39.59 27.49 40.20 25.59 38.49 - - 26.51 39.43 July 23.30 30.40 24.47 32.36 23.74 31.65 - - 23.83 31.47 August 22.40 28.20 23.04 28.69 22.38 29.58 - - 22.60 28.82 September 21.87 31.73 21.58 29.74 21.68 36.54 - - 21.71 32.67 October 20.27 33.62 19.56 31.69 20.76 32.44 - - 20.20 32.58 November 15.99 28.11 15.95 28.36 15.29 29.18 - - 15.74 28.55 December 12.66 25.67 13.02 26.44 12.19 25.60 - - 12.62 25.90

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 12 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

0 Fig. No. 3 2 Average monthly temperature ( C) at DamohSagar in different months of study period

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Table- 3: PERCENTAGE RELATIVE HUMIDITY AT SAGAR DURING STUDY PERIOD

Months Year Average 2001 2002 2003 2004 Min. Max. Min. Max. Min. Max. Min. Max. Min. Max. January - - 50.05 40.01 51.11 35.54 54.21 27.23 51.79 34.26 February - - 48.92 30.38 50.34 34.87 46.72 32.75 48.67 32.66 March - - 46.49 25.22 47.48 26.95 35.06 17.16 43.01 23.11 April - - 30.43 20.20 31.06 29.87 27.46 18.40 29.65 22.82 May - - 28.77 19.53 29.42 20.22 34.25 20.50 30.81 20.08 June 56.00 49.10 68.80 48.80 69.57 60.00 - - 64.79 52.63 July 91.20 78.23 86.46 69.44 90.47 70.00 - - 89.37 72.55 August 93.00 82.00 95.00 81.51 97.00 82.39 - - 95.00 81.96 September 80.36 62.21 98.00 70.31 95.83 80.45 - - 91.39 70.99 October 39.77 26.55 50.00 32.03 65.38 28.55 - - 51.71 29.04 November 53.85 44.14 48.33 38.55 53.37 20.21 - - 51.87 37.63 December 65.50 46.10 60.50 45.10 56.05 38.80 - - 60.68 43.33

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 13 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Fig. No. 4 3 Percentage relative humidity at DamohSagar during study period

100 90 80 70 60 50 40 30 20 10 Relative Humidity percentageRelativeHumidity 0 July May April June March August January October February November December September Months

2001 Mor. 2001 Eve 2002 Mor. 2002 Eve 2003 Mor. 2003 Eve 2004 Mor. 2004 Eve

RELATIVE HUMIDITY Humidity is an important factor. It is higher in rainy season, moderate in winter and least in summer season. Mean monthly records of relative humidity in morning and evening are given in table 3 and fig.3. DESCRIPTION OF SITES Seed collection of some medicinal plant species have been carried out in the adjoining forest of Sagar district. These are as follows: - 1. RAMANA: This forest is situated about 40 k.m. from Sagar on Jabalpur-Sagar road. Most dominant treee species are Tectona grandis, Terminalia, tomentosa, Lagerstroemia parviflora, Acacia catechu, Acacia leucophoelea, Anogeissus pendula, Butea monosperma, Cassia fistula, Gardenia latifolia, Flacourtia indica, 2. RAHATGARH: Rahatgarh is situated about 45 k.m. in east of Sagar on Bhopal- Sagar road. It consists of sandstone hills with an average height of 602 m. from sea level. This forest is dense and mixed in composition with well established mature trees of economically important timber value. The dominant tree species of this area are Tectona grandis, Madhuca indica, Diospyros malanoxylon, Lagerstoemia parviflora, Lannea coromandelica, Acacia catechu, Terminalia tomentosa, Holarrhena antidysentrica, Cassia fistula, Ziziphus xylopyrus etc.

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3. JAMGHAT: This forest is situated about 35 k.m. from Sagar on Sagar Jabalpur road. It lies on basalt rock. This forest is degraded Bushy habit of trees is common in this site. Terminalia arjuna, Terminalia tomenstosa, Tectoma grandis, Lagerstroemia parviflora, Butea monosperma, Wrightia tinctoria, Holarrhina antidysentrica, Carissa, spinarum are the dominant species of this site. 4. GOPALPUR: Gopalpur forest is situated about 60 k.m. in south of Sagar on Narsighpur-Sagar road. Tectona grandis, Lagerstroemia parviflora, Terminalia arjuna, Terminalia tomentosa, Madhuca indica, Anogeissus pendula, Lannea coromandelica are the dominant species of this site. 5. DHAMONI: Dhamoni forest is situated about 40 k.m. from Sagar on Lalitpur- Sagar road. This forest is dense and mixed in composition with well established mature trees of economically important timber value. The dominant tree species of this area are Diospyros melanoxylon, Lannea coromandelica, Acacia catechu, Terminalia tomentosa, Holarrhena antidysentrica, Cassia fistula, Ziziphus xylopyrus.

REFERENCES Kulczynski, S. (1937). The Study of Plant Communities, W.H. Freeman & Co., San Francisco. Rathore, J.S. (1968). The morphology and ecology of Bidi plant (Diospyrus melanoxylon Roxb.) Ph. D. Thesis, Univ. of Saugar, Sagar. Tiwari, P.K. (1994). Studies on seed biology and regeneration of certain forest tree species of Central India. Ph. D. Thesis. Dr. H. S. Gour Univ. Sagar. Wadia, D.N. (1948). Geology of India. Mc. Millan and Co. Ltd. London.

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Ethnobotanical Research Unfolds New Vistas For Rural Development of Bundelkhand

Soma Anand Saxena and A. P. Saxena Department of Botany & Industrial Microbiology Pt. Jawahar Lal Nehru P.G. College. BANDA (U.P.)

ABSTRACT Plants have been an integral part of Indian life and culture. There are profuse references of trees, shrubs, climbers and their flowers, fruits etc. in the folk literature of our country. A large number of wild plants being used by tribals and rurals of this country from ancient times for various purposes. The present paper deals with a brief account of ethnobotanical studies of Bundelkhand (U.P.). The ethnomedicinal uses of 18 plant species are incorpoated. These plant species are regarded as to be endangered in this area due to over exploitation. The method of conservation of these plants and its significance for rural development is also discussed. INTRODUCTION: On account of their great potentiality for the production of natural indigenous drugs, including various antimicrobial substances and because of the lack of any previous work on medicinal plants of Bundelkhand region, the attempts were made to study the plants used by the local inhabitants of this area for the treatment of common ailments including various infectious diseases. (Saxena, 1983) METHOD OF STUDY: The ethonobotanical investigations were carried out in selected localitites of Banda, Hamipur, Jhansi, Lalitpur and Mahoba districts of Uttar Pradesh. These areas are particularly rich in tribal and rural population as well as in wild plants. The data about the traditional use of medicinal plants for the treatment of various infectious and other important diseases were recorded by interviewing various tribals and other local inhabitants (Jain, 1981). Attempts were made to carry out ethnobotanical studies of kols, Gonds, Gujars, and Sahariya tribes of this region during 2002-05 and recorded various uses of about 98 species of plants.

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OBSERVATION: The observation have revealed the important ethnobotanical uses of plants suggested by the tribals and rurals for the purpose of food, fodder, medicine, veterinary, insecticides, tannin, dye, timber, fibre, oil, etc. During the course of investigation it was found that the tribals and more particularly Lodhis have been found to have a good deal of plants based information, practicing totems for curing the ailments. Besides, a good number of plants are found associated with religious sentiments of local inhabitants and some plants have become the part and parcel of local festivals. The tribals particularly Kols, Gonds and saharias appeared more conservative while some medicineman seemed proud of telling the secrets know to the them. An overall assessment has indicated that a large number of plants occupy foremost place in curing skin diseases, next in order coming those used in the treatment of diarrhoea and dysentery, fever, rheumatism, cuts and wounds etc. It was also recorded that in the wake of establishing themselves on one hand the rural people have exploited Kols and Gonds, the deforestation activities have caused destruction to their age old culture on the other. There has been a change in the outlook of tribals of Bundelkhand. The tribal section has been found maligned for many things like deforestation due to shifting cultivation and consequent loss of top soil which had led to creation of vast tracts of open lands most of which used agriculturally but the another vast tracts lying barren with bushy or no vegetation. Out of the plant species investigated for their ethnomedicinal importance as many as following 18 species are regarded as to be endangered. The ethnomedicinal uses of these plant species are discussed as below. 1. Alectra parasitica, A Rich var. Chitrakutensis, Rau. (Nirgundi) Air dried plants are mixed together with equal amount of seeds of Psoralia corylifolia and powdered. The mixture is recommended for oral and local administration for the total cure of leprosy and leucoderma. Loc. Chitrakut, Banda (U.P.) Ethm. Dist. Bundelkhand (Saxena & Vyas, 1983) 2. Anogeissus latifolia, Wall (Dhaura, Bakli) Decoction of the stem bark is used in Jaundice and disorders of stomach and liver. The wood is used for making agricultural implements. Leaves are used as a colouring material in local tannaries. Loc. Naghara, Hamirpur (U.P.) Ethn.Dist. Gujarat (Gopal and shah 1985), Maharashtra (Sharma and Lakshminarsimhan, 1986), U.P. (Khanna et.al., 1996) M.P. (Singh et.al.,. 1999).

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3. Anogeissus pendula, Edgw. The decoction of the powdered seeds is used in dysentery Loc. Syondhi, Hamirpur (U.P.) Eth. Dist. Bundelkhand. (Saxena & Vyas 1983).

4. Centella,asiatica (Linn.) Urban. (Brahmi) An infusion of the plant is used during dysentery and skin affections. It is said to be useful as a brain tonic. Loc. Charkhari, Hamirpur (U.P.) Eth. Dist. Bihar (Gupta, 1981), Orissa (Saxena et.al., 1981), nilgiris (Abraham 1981) U.P. (Singh et aI1980), Calcutta (Chakravarty, 1975), Nilgiris (Abraham, 1981), Assam (Hajra & Baishya, 1981) H.P. (Kapur,1986), T.N. (Banergee & Banergee 1986).

5. Chloraphytum tuberosum, Baker (Safed-musari) The powdered tubers are used for the treatment of leucorrhoea. Loc. Syondhi, Hamirpur (U.P.) Ethn. dist. Bundelkhand (Saxena & Vyas, 1983), Maharashtra & Goa (Vartak 1981) M.P. (Roy & Chaturvedi 1987)

6. Curculigo orchioides, Gaertn. (Kali Singhia) The powdered root is used during diarrhoea and dysentery. Fresh root is cut and placed on the spot of scorpion-sting to relieve the pain. Loc. Pachora, Lalitpur (U.P.) Ethn.dist. Bihar (Tarafder and chaudhuri, 1981 ),Orissa (Saxena et.al., 1981), Andhra pradesh and Orissa (Jain et.al., 1973, Raju 1995), M.P. (Jain, 1965) Maharashtra (Mudaliar et.al., 1987), Rajasthan (Singh & Pandey 1996).

7. Elytraria acaulis. Linn. (Sahastra musari) Fresh leaves are pounded into paste and applied externally on wounds and nail diseases. The powdered roots mixed with leaves of tobacco are used as poultice to the sores of cattles. Loc. Kalinger, Banda (D.P.) Ethn. Dist. Bundelkhand (Saxena and Vyas, 1981), M.P. (Panuli & Maheshwari, 1996).

8. Gloriosa .superba, Linn. (Kalihari) The root is purified by keeping it in the milk for a week, it is then dried and powdered. The Bowder mixed with Gram's meal is recommended for oral administration to the cattles during rheumatism. Loc. Banda (D.P.) Ethn. Dist. Bihar (Trafder and chaudhuri, 1981), Orissa, (Saxena et.al., 1981) (Singh et.al., 1980), Rajasthan (Singh & Pandey, 1996) M.P. (Samvatsar & Diwanji 1999).

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9. Helicteres isora. Linn. (Marorphali) Powdered fruits are recommended for oral administration along with water or small quantity of sugar during griping of bowels or stomachache. Loc. Kuchhechha, Hamirpur (D.P.) Ethn. dist. Orissa (Saxena et.al., 1981), Gujarat (Joshi et.al., 1980), M.P. (Jain, 1965) Maharastra (Shah et.al., 1983), Bihar (Girach & Aminuddin, 1995).

10. Hemidesmus indicus. Br. (Jangali sariva) The aqueous extract of the root is provided for the treatment of tonsilitis in children. Loc. Naghara, Hamirpur (D.P.) Ethn. Dist. Bihar (Gupta 1981), Orissa (Saxena et.al., 1981), Gujarat (Joshi et.al.,., 1980), Andhra Pradesh and orissa (Jain; et.al., 1973), M.P. (Jain, 1965), Rajasthan (Singh & Pandey 1996).

11. Holarrhena antidysenterica, Wall (Sundari) The seeds are slightly warmed and powdered. The powder is used for the, treatment of dysentery. An infusion of stem bark is also recommended for oral administration in dysentery. Loc. Chitrakut, Banda (U.P.) Ethn. Dist Assam (Hajra & Baishya, 1981), Bihar (Gupta, 1081), Orissa (Saxena et.al., 1981), Gujarat (Joshi et.al., 1980), U.P. (Maheshwari et.al., 1980) M.P. (Jain, 1965, Saxena & Patnaik 2000), Calcultta (Chakravarty 1975).

12. Pedalium murex, Linn. (Badi Gokharu) The dried and powdered fruits are used for the treatment of glycosuria and impotence. Loc. Syondhi, Hamirpur (U.P.) Ethn. dist. Bundelkhand (Saxena & Vyas, 1983), Maharashtra (Shah et.al., 1983), Rajasthan (Das, 1997).

13. Phoenix sylvestris, Roxb. (Khajur) Brooms, baskets and toys are prepared with the leaves. Sap from the stem apex, 'Nira' is useful in asthma. It is also used for preparation of guglery "Gur" Loc. Sugira, Hamirpur (U.P.) Ethn. Dist. Bundelkhand, U.P. (Bajpayee & Dixit 1996), M.P. (Maheshwari 1996), Rajasthan (Das 1997).

14. Plumbago, zeylanica, Linn (Chitrak) Decoction of the root is used during fever. Loc. Chitrakut, Banda (U.P.) Ethn. Dist. Bundelkhand (Saxena and Vyas, 1981), Bihar (Gupta, 1981), Orissa (Saxena et.al., 1981), Andhra Pradesh and orissa (Jain et.al., 1973), M.P. (Jain, 1965), Assam (Tiwari, et.al., 1980), Meghalaya (Neogi et.al., 1989) Rajasthan (Das, 1997).

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15. Psoralea corylifolia, Linn (Bakuchi) Seeds are powdered and mixed with the equal quantity of dry and powdered plant of Eclipta alba. The whole powder is dipped in water for 24 Hours and filtered. The filtrate is recommended for oral administration for the treatment of leprosy. Leaves grinded with mustard oil and applied externally to itch. Loc. Gorahri, Hamirpur (U.P.) Ethn. Dist. Calcutta (Chakravarty, 1975) Rajasthan (Sharma, 1990), M.P. (Samaratsar & Diwanji 1996)

16. Selaginella bryopteris (L) Baker. (Kamraj) The air dried plants are powdered and boiled with mustard oil, the oil is filtered and rubbed on the body of the child suffering from marasmus. Loc. Chitrakut, Banda (U.P.) Ethn. dist. Bundelkhand (Saxena & Vyas 1983), U.P. (Singh et.al., 1996)

17. Trichodesma,indicum, Br. (Ondhelu) Decoction of the plant is used as brain tonic. Expressed Juice of the leaves is applied for the treatment of itch and eczema. Loc. Syondhi, Hamirpur (U.P.) Ethn. Dist. Bundelkhand. (Saxena, 1983)

18. Vitis quadrangularis, Wall (Hadjoru) Fresh stem pounded into paste and the poultice is used for external application.on bone fracture. Loc. Chitrakut, Banda (U.P.) Ethn. Dist. - Bundelkhand (Saxena, 1983)

DISCUSSION: The ethnobotanical survey of Bundelkhand (U.P.) has revealed the richness of folklores which require special attention and remedial measures for their conservation. The conservation of these living resourses reflects three specific objectives: to maintain essential ecological processes and life support system. to preserve ethonobotanical diversity and to sensure that any utilisation of species and ecosystem is sustainable. Conservation, therefore is expected to make important contributions to social and economic development of the backward region. Two different forms of conservation can be adopted to preserve the plant wealth: in-Situ conservation and ex-Situ Conservation. (1) in-Situ Conservation/ Agro Forestry in-Situ approach is the ideal system of conservation because of the fact that not much diversity can be conserved outside the centres of diversity. It is the conservtion of genetic resoruces through their maintenance within natural or even man made ecosystem in which they occur. The best way to achieve this is the promotion of cultivation and protection to all plants in general and endan- gered ones in particular. Being an agriculture based area the cultivation of fibre yielding, dye yielding, oil yielding and some prominant medicinal plants will be

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beneficial not only from conservation point of view but from economic point of view as well. For this purpose the following plants are proposed to be used.

(2) Asparagus racemosus, Willd (3) Aloe barbadensis, Mill (4) Chlorophytum tuberosum, Baker. (5) Holarrhena antidysenterica, Wall Cat. (6) Ocimum sp. (7) Pedalium murex, Linn. (8) Plumbago zeylanica, Linn (9) Psoralea corylifolia, Linn. (10) Ricinus communis, Linn (11) Tinospora cordifolia Miers.

(2) ex- situ Conservation : It is the conservation of a living being outside its habitat by perpetuating sample population in genetic resource centres, botanical gardens, culture collections etc. or in the form of gene-pools, germplasm banks for seeds, pollen, cells etc. It is the safest and cheapest of life processes which are reduced to the minimum level. Establishment of culture collection centres and regional ethonobotanical gardens in this area will be a great service to mankind via protecting plant wealth and the ethnic knowledge related with it. The endagered plants listed above should be given due attention on this score.

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7. Gopal, G.V. and G.L.Shah, 1985. : Some Folk Medicinal Plants used for Jaundice in Gujarat- India. Jour. Rec. Edu. Ind. Med. July-Dec., 45-49.

8. Gupta, S.P. 1981 : Native Medicinal uses of Plants by the Asus of NetarhatPlateau (Bihar) In : Glimpses of Indian Ethnobotany : 218- 231. Oxford & IBH Pub. Co., New Delhi.

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12. Joshi, M.C. M.B. Patel and 1980 : Some folk Medicines of Dangs, Gujarat R.J Mehta State, Bull. Medico- ethnobotanical Research. 1(1) : 8-24.

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13. Kapur, S.K. 1986 : Vegetable Raw Material Resources of Kangra Valley (Himachal Pradesh), J. Econ. Tax, Bot., 8 (1) : 65-76.

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23. Saxena A.P. 1983 : Studies on medicinal plants of Bundelkhand region with special reference to their anti-microbial activity. Ph. D Thesis approved by Dr. H.S. Gaur University Sagar (M.P.)

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34. Singh, V. and R. P. Pandey, 1996 : Ethno-Medicinal Plants used for General & Gynaecological diseases in Rajasthan (India). J. Econ. Taxon. Bot. 12: 154-165.

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Evaluation of fungicides and relation with weather conditions in association of Alternaria alternata with Ashwagandha

N.P. Mandloi* , S.K. Arsia** and A. N. Rai* *Department of Botany Dr. Hari Singh Gour Central University, Sagar (M.P.) India **Department of Plant Pathology RVSKVV Gwalior (MP)

ABSTRACT Five different fungicides were evaluated against Alternaria alternata on Ashwagandha seeds revealed that maximum germination was recorded in thiram followed by Mancozeb, Chlorothalonl, Copper oxychloride, and Carbendazim with 55,49,48,47 and 44 per cent, respectively. The germination in treated seeds was significantly superior over control. Correlation of fortnightly progress of the development of leaf blight on Ashwagandha showed a positive correlation with maximum temperature and minimum temperature. However, a positive correlation was observed with relative humidity. This indicates that lower maximum temperature, low minimum temperature with higher relative humidity favour the development of the disease in the field. These conditions normally presents during 1st and 2nd week of February when maximum disease was observed . Keywords : Ashwagandha /Alternaria alternata /fungicides /weather conditions Introduction: Ashwagandha (Withania somnifera L. Dunal.) is an important tropical medicinal plant of India and belongs to family Solanaceae. The plant is native of drier parts of the country, ascending upto 1700 m in the Himalayas. It is found sporadic to scattered in Guna, Hamirpur, Kullu (Bhunter) Kangra, Mandi, Bilaspur (Bhararighat), Solan and Sirmour districts at Himachal Pradsh upto an, altitude of 1400m (Chauhan, N.S.1965) It is commonly used in Ayurveda and other traditional systems of medicine (Bhargava and Singh, 1978). Ashwagandha is also known as Indian ginseng, cluster or winter cherry. Ashwagandha is cultivated in an area of about 4000 ha in India. Ashwagandha is a flok remedy for diseases viz., adenopathy, anthrax, arthrits, erysipelas, fever, hypertension, inflammation, marasmus, housea, piles, proctitis, ringworm, scabies, smallpox, sores, syphilis, tuberculosis and typhoid. Die-back and leaf blight diseases caused by Alternaria alternata cause significant damage to the crop. The plant population is drastically reduced resulting in reduced root yield (Prajapati, 2003). In Madhya Pradesh it is cultivated mainly in Neemuch, Chhindwara, Mandsaur, Seoni, Katni, Shahdol, Dewas, Hoshangabad, Jabalpur and Dindori districts with a production of 15.46 q/ha (Anonymous, 2003). This crop is vevy important as

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 26 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 medicine no sufficiant work on various aspect of leaf blight disease has been carried out therefore, an attempts has been made to study the various aspects of this disease. Material and Method The association of Alternaria alternata with the seeds of Withania somnifera was detected using standard blotter method (ISTA, 1976). Pre-sterilized four blotters of Petriplate size were cut and dipped in sterilized water and placed in sterilized Petriplates to supply moisture to the seeds for germination. Surface sterilization of seeds was done with mercuric chloride solution (1:1000) and then washing in sterilized water thrice. Twenty five seeds were placed in each Petriplate in such a way that fourteen seeds formed the outer circle and ten seeds formed second circle and one was placed in the center. In all, 400 seeds were incubated for each of the 4 replications in an incubator at 240C. Four lots of un-sterilized seeds were kept along with the sterilized seeds to serve as control. Studies on the incidence of leaf blight were undertaken from January 10 to February 26,2005 i.e. from disease appearance till the end of disease development in the farmers fields by recording disease development. The weather data, temperature and relative humidity was then correlated with disease incidence. The data analyzed statistically and correlation coefficient was calculated. Result and discussion Alternaria alternata, was found associated with Ashwagandha seeds to the extent of 55 per cent as detected by blotter paper method. Such seeds were observed to be shriveled with less seed weight. Fungicides tested resulted in better efficacy in reduction of seed borne fungi and improvement in germination by thiram(55percent), mancozeb(49percent) and chlorothalonl(44 percent). A large number of fungi have been reported to be associated with seeds of medicinal crops species of Aspergillus, Penicillium, Mucor and Curvularia lunata make their apperance on a variety of crop seeds with there parasitic and saprophytic importance. Gupta and Rana (1981) detected Aspergillus spp., and C. lunata from tomato, chilli and brinjal seeds. Dwivedi et al. (1982) isolated 45 fungi from seeds of various medicinal plants which not only deteriorate the seed quality but also reduce the alkaloid contents. Alternaria alternata is a well known pathogen of several crops with its seed borne nature of transmission. Association of this fungus with Ashwagandha seeds is in conformity with its seed borne nature. Earlier it has also been reported from seeds of tomato, chilli and brinjal (Gupta and Rana, 1981); soybean (Morsy et al., 1985) and Indian mustard (Rani and Agrawal, 1995). The investigation at fortnightly interval on the progress of leaf blight of Ashwagandha revealed the maximum incidence was noticed in the month of February.

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During this period the relative humidity was noted to be 65.5 percent with maximum and minimum temperature 28.6 and 14.5 0C, respectively .The correlation studies suggested that a positive correlation of disease development was observed with maximum and minimum temperatures. However, there was a positive correlation with relative humidity. This indicates that lower maximum and minimum temperatures and higher relative humidity favour the development of the disease in the field. Lucic (1967) reported the optimum temperature for the development of Alternaria alternata causing black spot of tomato as 26 0C. Gemawat and. Prasad (1972) reported that development of Alternaria blight in cumin requires 90 per cent and above relative humidity for about 3 days and temperature between 25 and 28 0C. Theses observations thus indicate that lower temperature and higher humidity favour the development of disease caused by Alternaria alternata, on the crops. References: Chauhan, N.S. (1965). Descriptive Profile of Plants. Medicinal and Aromatic Plants of Himachal Pradesh. pp. 439-442. Bhargava, N.C. and O.P. Singh (1978). Fortege an indigenous drug in common sexual disorders in males mediscope. 21 : 140-141. Prajapati, N.D., S.S. Purohit, A.K. Sharma and T. Sharma (2003). A Hand Book of Medicinal Plants, a complete source book. Agrobios (India), pp. 108-109. Anonymous (2003). Cultivation practices of some commercial important medicinal plants. National Plant Board, Gupta, P.C. and V.P. Rana (1981). Effect of culture filtrate of some seed mycoflora on seed Germination of Solanaceous crops. Seed Res.,9 (2): 192-193. Dwivedi, D.K., S.N. Bhargava and D.N. Shukla (1982). Fungi isolated from seeds of spices. Natl. Acad. Sci. Lett., 5(3):75-79. Morsy, A.A., A.F. Sahab; M.M. Diab and A. Nofalin (1985). Determining seed health of soybean by the effect of seed borne fungi on germination invasion and occurrence in culture. Egyptian J. Phytopath., 14 :75-82. Rani, P. and A. Agrawal (1995). Effect of fungicides on seed mycoflora and seed germination on mustard. Adv-in-Plant-Sci.,8(2):342-344. Lucic, Solbodanka (1967). Contribution to the study of Alternaria tenuis the casual agent of black spot of tomato. Zast. Bilja, 18:161-168. Gemawat, P.D. and N. Prasad (1972). Epidemiological studies on Alternaria blight of Cuminum cyminum. Indian J. Mycol. Pl. Pathol., 2:65-74. Table :- 1 Association of Alternaria alternata with the seeds of Ashwagandha

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Treatment Concentration % *Mean *Germination association of (%) A.alternata (%) Carbendazim 0.1 8 44 Thiram 0.3 6 55 Chlorothalonl 0.2 9 48 Mancozeb 0.3 7 49 Copper oxychloride 0.3 11 47 Control 0.0 15 41 S.Em (+) = 2.11 3.28 C.D. (P=0.05%) = 4.39 6.83 Figures in parentheses angular transformed values . *Mean of four replications of 100 seeds.

Table-2 Incidence of leaf blight of Ashwagandha at fortnightly interval in relation to weather parameters Month/ Year Max. Temp. Min. Temp. R.H. (%) Incidence of (0C) (0C) leaf blight (%) 10 Jan,2005 23.5 6.8 65.5 4.24 25Jan,2005 23.9 10.3 68.5 6.92 09Feb,2005 28.6 14.5 65.5 8.721 24Feb,2005 25.5 10.8 61.0 6.44 Correlation +0.84 +0.98 +0.096 coefficient

Incidence of leaf blight (%) Temp. (0C) R.H.(%)

100 90 80 70 60 50 Date 40 30 20 10 0 10 Jan,2005 25Jan,2005 09Feb,2005 24Feb,2005

Locations Fig.2: Incidence of leaf blight of Ashwagandha at fortnightly interval in relation to weather parameters

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Assessment of biochemical tests for seed viability and vigour in a multipurpose forest tree: Butea monosperma (lam.) Taub. V.K. Yadav and P.K. Khare* Department of Botany D.V. College, Orai-285001 INDIA *Department of Botany, Dr. H.S. Gour University, Sagar-470 003 India

ABSTRACT Tetrazolium (TZ) and Indigo Carmine (IC) topographical staining are important and common seed viability tests. However conducting and interpretation of these tests has proven to be extremely subjective and tedious. The paper deals with the comparison of two viability testing methods viz., Tetrazolium and Indigo Carmine staining in seeds of Butea monosperma. Results indicate that both the methods are reliable for testing the seeds in this species. On the basis of the present study it has been concluded that the indigo carmine staining is easy, less time consuming and economical as well as the stain can be stored for a longer period, than tetrazolium staining method. Indigo carmine staining method is recommended for seed testing. INTRODUCTION Knowledge of seed viability is the first and foremost requirement prior to raising the plants and for storage of seeds. Before the seeds are sown or placed in storage, the viability is measured to determine whether the seeds have potential for germination and survival in future. During storage, the seed samples are regularly monitored Knowledge of seed viability is the first and foremost requirement prior to raising the plants and for storage of seeds. Before the seeds are sown or placed in storage, the viability is measured to determine whether the seeds have potential for germination and survival in future. During storage, the seed samples are regularly monitored for their viability. For this purpose, besides germination test biochemical tests are recommended as quick test for testing seed viability .Kuhn and Jerchel (1941) first introduced the tetrazolium test for seed viability. It was further used by Lakon (1942) for topographical evaluation of viability by staining embryo. Later on several workers have used this method for testing seed viability in vegetables, cereals and forest tree species (Gopal and Thapliyal, 1969; Agarwal and Kaur,1975; Kandya and Babeley, 1984; Yadav et al. 1992; Yadav and Khare, 2003). Tetrazolium test has also been included in the rules for seed testing by ISTA (1976, 1985). Another biochemical test of seed viability, the indigo carmine test, was introduced by Neljubove (1925) but it remained unused for some unknown reasons. This test has not been included in the rules of seed testin by ISTA although a number of workers have established its

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 30 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 reliability (Kamara, 1972). There are various workers (Simak and Kamara, 1963; Kamara, 1972; Yadav et al., 1988, 1992; 2003). The test needs more recommendations for its inclusion after its validity and performance in large and varied species. Butea monosperma (Lam..) Taub (Fabaceae) is a moderate sized deciduous tree with irregular branches and crooked trunk of the Indian sub continent (Troup, 1921). It is an important species that yield several non timber forest produce such as water soluble dye, lac, resin leaves for plates, thatching, roots for fibers etc. (Anon, 1988). The tree with beautiful orange flowers in leafless canopy during the summer season is aptly described as the flame of the forest and attract a large number of bees and birds (Anon.1988, 1994). In the present study tetrazoliun and indigo carmine tests were performed for testing seed viability in Butea monosperma. These biochemical l tests were compared for their reliability in testing the viability and vigour in B.monosperma seeds.

Materials and Methods Seeds of B.monosperma were collected from tropical dry deciduous forests of Sagar. Seeds (100 x 4) for each test were soaked in water for 6 to 8 hours at room temperature. A lot of extra seeds was also kept separately to replace those could be damaged during the process. Seed coats of imbibed seeds were removed and the seeds were cut longitudinally into two halves and were kept in 0.1% solution of 2,3,5- triphenyle tetrazoliun chloride (pH 6.5-7.0) at 30 C in dark (ISTA,1985). After incubation (6-8 hours), seeds were immediately washed with water 2-3 times to drain the excess dye. Seeds were then place on glass plates for further evaluation. Similarly seeds were immersed in 0.1% solution of indigo carmine and incubated for 2 hours. After incubation the seeds were rinsed with water to remove the excess indigo carmine dye. Topographical evaluation of stained seeds was done as follows: (1) Tetrazolium Staining: Seeds were placed on glazed glass plates and observed under a stereo microscope for evaluation as per the rule 6.5 2A (ISTA, 1985). All the essential structures such as meristem and embryo were examined for staining. Seeds with weakly stained embryo with non stained other parts were considered as non viable (Leadem, 1984). On the basis of degree of staining and staining patterns, seeds were classified into different categories. (2) Indigo Carmine Staining: Seeds were arranged in row on a white plate for examination of degree of coloration under stereo microscope. On the basis of non coloration of essential parts of the seeds, five viability categories were recognized (Baldwin, 1942).

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For seed germination 4x100 seeds were placed on moistened sterilized filter papers in a seed germination incubator at 28+2 C. Observations were made daily up to 28 days and radical emergence was taken as criterion for germination (ISTA,1985). Results and Discussion After a careful of staining pattern of tetrazolium and indigo carmine staining, seeds were classified in to different staining categories (Table 1 and 2). Stained seeds were also classified in to vigour classes on the basis of their staining patterns. In case of tetrazoliun staining, seeds stained fully including both embryo and cotyledons were classified in category 1. In all eight staining categories were made and percentage of seeds belonging to a category w re compared with actual laboratory and field germination. Comparison was also made by totaling percentage values from category 1 down to 6 till the value near the germination was obtained. Student ‘t’ test was performed between total of different categories and percentage laboratory and field germination. It is evident from the data that staining categories up to 4 can be considered as viable as there is no significant difference in ‘t’ value between the viability and germination (Table 3 ). Similarly, indigo carmine staining of seeds was also interpreted on the basis of staining pattern. Seeds without staining were classified in most viable category and other categories were made on the basis of increasing staining patterns. Results of student ‘t’ test showed that viable categories include from 1 to 4. The evaluation of vigour in seeds was also done on the basis of staining pattern. In case of tetrazoliun staining, seeds with deeply stained embryo have been classified as fast vigour seeds and vise-versa in case of indigo carmine staining. The 2,3,5-triphenyl tetrazoliun chloride is reduced by terminal oxidase system in living plant tissue from a colourless solution to red , water insoluble farmazon compound which is precipitated in the living cells, while in dead cells no reaction takes place hence no colour develops (Kuhn and Jerchel, 1941 ). On the other hand Neljubove (1925) reported that indigo carmine stains only dead cells or dying tissue of embryo readily but leaves the living tissue unstained. The comparison of germinability of five samples of Pinus sylvestris seeds tested with tetrazoliun chloride and X-ray contrast method using the germination on the basis of stained embryo alone give higher values (Simak and Kamara,1963 ). Kamara (1972) investigated the results of germination in seeds of Pinus sylvestris and Picea abies by indigo carmine and X-ray contrast methods using the value of germination as standard, reported satisfactory results of seed viability.

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It is evident from the results that both the staining test methods are reliable for estimating viability correctly in the seeds of Butea monosperma Similar results have been obtained by Yadav et al. (1988, 1992, 2003)in seeds of Chloroxylon sweitinia, Dendrocalamus strictus and Bauhinia variegata. They have determined the viability of seeds by tetrazoliun chloride and indigo carmine staining. Comparison of these two viability tests envisaged that the indigo carmine staining test is less time consuming, economical and the stain can be stored without damage. The present study showed that the viability of Butea monosperma seeds can be predicted on the basis of tetrazoliun and indigo carmine staining tests and the latter test may be incorporated in the rules of seed testing. REFERENCES Agrawal, P.K. and Kaur,S. 1975. Standardrization of TZ test for Ragi (Eleusine ciracassa ) seeds. Seed Sci. and Technol. 3:565-568.

Anon., 1988. Butea monosperma (Lam.) Taub. The Wealth if Inia –raw material. Revised Vol. II B . C.S.I.R., New Delhi. Anon., 1994. Indian medicinal plants: a compendium of 500 species.Vol. 1 Madras. Orient and Longman Ltd. Baldwin, H.I. 1942. Forest tree seeds of North temperate regions with special reference to America, Chronica Botanica Co., Waltharn Mass, U.S.A. Gopal,M. and Thapaliyal, R.C. 1969. Topographical TZ test for Indian tree seeds. Van Vigyan, 7:37-45. I.S.T.A., 1976. International Rules for Seed Testing. Seed Sci. and Technol., 4 : 3- 177. I.S.T.A., 1985. International Rules for Seed Testing. Seed Sci. and Technol., 13 : 300- 520. Kamara, S.K. 1972. Comparative studies on germinability of Pinus sylvestris and Picea abies seeds by indigo carmine and X- ray contrast methods. Studia Forestalia, 99 : 1-3. Kandya, A.K. and Babeley, G.S. 1984. Tetrazolium colouring and index for tests of viability and vigour of some tropical forest tree seeds. Proc. IUFRO Symposium on seed problems. Curitiba, Brazil,495-501. Kuhn, R. and Jerchel, D. 1941. Uber Investsei ferm VIII : Reduclion von tetrazolium salze durch Beklerien,grarende. Hete and Keimende Samen. Ber Deutsch Chem. Ges., 74 : 949-952.

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Lakon, S. 1942. Topographischer Nachweis der keimfahigkeit der Gertrei defruchte durch tetrazoliun salze. Ber Deusch. Bot. Ges. Co. 299-305. Neljubove, D.N. 1925. Uber die methoden der Bestimmung der keimfahigkeit ohne keimprufung. . Ann. Ess. Semences. Leningrad 4 :31-35. Simak, M. and Kamara, S.K. 1963. Comparative studies on Scot pine seed germination with tetrazolium and X-ray contrast methods. Proc. Int. Seed Test. Assoc. 28 :3- 18. Troup, R.S. 1921. The Silviculture of Indian Trees. Vol. 1 Clarendon Press, Oxford. Yadav, V.K., Khare, P.K. and Mishra, G.P. 1988. Seed viability test in Chloroxylon sweitinia. J.Ind. Bot. Soc., 67: 306-308. Yadav, V.K., Khare, P.K. and Mishra, G. P. 1992. Staining methods for testing viability in seeds of Dendrocalamus strictus nees. A comparative study. Range Mgmt. and Agroforestry, 13 (1) : 49-56. Yadav, V.K. and Khare, P.K. 2003. Comparative study of topographical staining methods for testing viability in seeds of Bauhinia variegate Linn. Flora and Fauna, 9 : 41-44.

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Table 1. Various categories of tetrazolium staining pattern in seeds of B. monospema with viability and vigour classes (Seed germination– 49.25%) Frequency Catagory Viability Vigour Staining pattern (%)

Embryo and cotyledon 1. 24.5 + Very fast completely stained

Embryo and cotyledon stained except a small 2. 2.25 + Fast portion of cotyledon opposite to redicle end

Embryo and cotyledon 3. stained except periphery 6.0 + Slow of cotyledon

Embryo and cotyledon 4. stained except redicle 2.25 + Very slow tip

Embryo and less than 5. 50% cotyledon 17.5 - No growth unstained

Embryo unstained with 6. 12.5 - No growth cotyledon light stained

Embryo unstained with 7. 8.25 - No growth cotyledon light stained.

Embryo and cotyledon 8. 26.75 - No growth unstained

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Table 2. Various categories of indigo carmine stainining pattern .in seeds of B. monospema with viability and vigour classes (Seed germination– 49.25%) Catagory Frequency Viability Vigour Staining pattern (%) 1. Embryo and cotyledon 29.5 + Very fast unstained

2. Embryo and cotyledon 1.25 + Fast unstained except a portion of cotyledon

3. Periphery of cotyledon 7.25 + Slow stained

4. Redicle tip stained 2.00 + Very slow

5. Embryo and less than 12.00 - No growth 50% cotyledon stained

6. Cotyledon stained 14.5 - No growth dotted

7. Embryo stained but 11.25 - No growth cotyledon unstained.

8. Embryo and cotyledon 22.25 - No growth stained

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Table: 3 : Summary of Student 't' test between different seed categories stained with tetrazolium and indigocarmine, and germination in Butea

monosperma (t1- seed categories Vs laboratory germination, t2- seed categories VS field germination, df-3). Tetrazolium staining:

Source of variation Sum of Source T1 T2 1 & 2 26.75 10.00** 5.18* 1, 2 & 3 32.75 6.88** 1.90NS 1, 2, 3 & 4 35.00 6.36** 1.03NS 1, 2, 3, 4 & 5 52.50 2. 16NS 5.15*

1, 2, 3, 4, 5 & 6 65.00 2. 66NS 7.35**

Indigo carmine staining

Source of variation Sum of Source T1 T2

1 & 2 30.75 4.35* 3.38* 1, 2 & 3 38.00 2.70NS 0.0883NS 1, 2, 3 & 4 40.00 2.6 7NS 0.8823NS 1, 2, 3, 4 & 5 52.00 0.5238NS 4.10*

1, 2, 3, 4, 5 & 6 66.50 3.44* 6.62**

(Significance: *-0.05, **-0.01, NS-not-significant)

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Control of growth of Penicillium citrinum by certain medicinal plant extracts Supriya Gupta*, Anand Rajoria, Ravindra Singh Thakur and D. C. Atri Laboratory of microbiology and Plant Pathology Department of Botany School of Biological and Chemical Sciences Dr. H. S. Gour Vishwavidyalaya, Sagar (M.P.) 470003, India. ABSTRACT: Attempts were made to disclose the potentials of 10 medicinal plant extracts to inhibit the growth of Penicillium citrinum. For the purpose, leaf extracts of Azadiracta indica, Eucalyptus globusa, Datura alba, Mentha piperta, Osimum sanctum, Andographis paniculata, Azolla, Cajanus cajan, aerial part extract of Silybum marianum, and seed extract of Swietenia mahagoni were used. Their antifungal potential was tested under in vitro conditions at five different concentrations, viz, 3, 5, 10, 20 and 50 mg/ml. Surprisingly all the test medicinal plant extracts failed to show any striking antifungal activity, rather enhancement in fungal growth was noticed in case of A. indica and C. cajan extracts at 50 mg/ml concentration. Need to exploit additional plants is urged. Key words: Antimicrobial, Penicillium citrinum, medicinal plant extracts INTRODUCTION Synthetic drugs are currently being used as primary means for the control of diseases caused by fungi. However, the alternative control methods are needed because of the negative public perceptions about the use of synthetic medicines, resistance of fungal pathogens and high development cost. The uses of plant-derived products as disease control agents have been studied, since they tend to have low mammalian toxicity, less environmental effects and wide public acceptance (Lee et al., 2007). Nature has bestowed on us a very rich botanical wealth and diverse types of plants grow in different parts of the country conclusively referred to as medicinal plants. Medicinal plants represent an ample source of antimicrobial agents (Mahesh & Satish, 2008) and are already being used in traditional medicine (Mann et al., 2008). The potential of higher plants as sources for new antimicrobials is still largely unexplored. Among the estimated 250,000-500,000 plant species, only a small percentage has been investigated phytochemically and the fraction submitted to biological or pharmacological screening is even smaller. The utilization of plant materials to protect field crops and stored commodities against microbial attack has a long history. Considering the vast potentiality of medicinal plants as source of antimicrobial drugs, the present research work was designed to explore the anti Penicillinic potential of 10 medicinal plants, viz, Azadiracta indica, Eucalyptus globusa, Datura alba,

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Mentha piperta, Osimum sanctum, Andographis paniculata, Azolla, Cajanus cajan, Silybum marianum and Swietenia mahagoni. MATERIALS AND METHODS Collection of plant materials The leaves of medicinal plants viz: Azadiracta indica, Eucalyptus globusa, Datura alba, Mentha piperta, Osimum sanctum, Andographis paniculata, Azolla, Cajanus cajan, aerial part of Silybum marianum, and seeds of Swietenia mahagoni were collected in sterile polythene bags from the Botanical garden, Department of Botany, Dr. H. S. Gour University, Sagar and authenticated in the department. The standard toxigenic strain of P. citrinum (SOPP no. 1910) was obtained from Vellore Institute of Technology (VIT), Chennai. Preparation of plant extracts Leaves, aerial parts and seeds of the medicinal plants were shade dried, and powdered. The powdered plant materials were defatted with petroleum ether. 10.0 g of defatted powder was mixed with 150.0 ml of 70% methanol, kept at 50oC on water bath for 2 hours and then shaken for 72 hours. The extracts were then filtered and evaporated to complete dryness. The residues were collected and stored in refrigerator for further use (Mossini et al, 2004). Phytochemical Analyses The phytochemical tests were performed according to the methods suggested by Tiwari et al 2011.

TreasePreparation and Evans, of different 1983 and concentrations of plant extracts Different aqueous concentrations viz, 3.0, 5.0, 10.0, 20.0 and 50.0 mg/ml of plant extracts were made from the viscous residue in appendorff tube. ANTIFUNGAL ACTIVITY OF MEDICINAL PLANT EXTRACTS Preparation of Spore Suspension For experimental purpose a loopful of spores of P.citrinum were suspended into 10.0 ml of sterile distilled water and mixed thoroughly by vigorous shaking. Spore count was measured by placing 10.0 μl of the spore suspension on haemocytometer. The concentration of the spores was maintained to 105 – 106 cfu/ml. Effect on the growth of P.citrinum Effect on the growth of P.citrinum was measured as dry mycelial weight. 1.0 ml each of the extract dilutions were added aseptically to 50.0 ml YES broth contained in 150 ml borosil flasks after autoclaving at 1210C and 15 lbs pressure and then 0.5 ml of the

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 39 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 spore suspension was added. In control 1.0 ml sterile distilled water was used instead of plant extract. The flasks were then incubated at 280C for 10 days. After that, the broth was filtered through whatmann filter paper no.1. The mycelium was then dried on pre-weighed filter paper in oven at 600C for 24 hours. Dry mycelial weight was calculated and the percentage inhibition of mycelial growth over control was calculated as under (Shrivastava and Atri, 1998).

RESULTS AND DISUSSION As depicted in table-1, the phytochemical analysis of medicinal plants was done to ascertain the presence of active phytochemicals that play crucial rule as antifungal agents. The analysis revealed the presence of terpenoids in all the experimental plants. Anthraquinones were absent in all the plants. On the contrary cardioglycosides, alkaloids, flavanoides, tannins, saponins and steroids were found in maximum plant species with few exceptions. In all 5 concentrations, viz, 3, 5, 10, 20 and 50 mg/ml of each of the medicinal plant extracts were tested to evaluate their anti Penicillinic activity. All the test medicinal plant extracts did not possess any striking antifungal activity, rather enhancement in fungal growth was noticed when media was supplemented with A.indica (-1.805%) and C.cajan (-2.076%) extracts at 50 mg/ml concentration. Though, the plant extracts proved ineffective against mycelial growth, yet sporulation was inhibited in case of A.indica, C.cajan and Azolla. This observation is also supported by findings of Simone et al., 2009. Maximum antifungal activity was exhibited by E.globusa (9.070%) followed by S.mahagoni (8.62%) at 50 mg/ml concentration (Table 2, Graph 1). Rest all other plant extracts did not show any acceptable mycelial inhibition. As far as effect on mycelial growth is concerned results were discouraging as none of the medicinal plant extracts were effective in constraining the growth of P.citrinum more than 10% in terms of dry mycelial weight. Basically the reason behind the antifungal activity of the medicinal plants is believed to be due to the presence of secondary metabolites which act either individually or in concert (Simone et al., 2009; Parekh et al., 2005 and Joshi et al., 2011). Author is at loss in tackling the debacle (poor antifungal activity) witnessed as failure in achieving the level of antifungal activity in the present study which could be reasonably accommodated. Reasons are beyond comprehension. Perhaps more plant extracts could be exploited in the study and rigorous evidence fetched at the level of working of secondary metabolite substances, which could not be pursued due to lack of proper lab facilities that my laboratory could offer.

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Table 2: Effect of plant extracts on the growth of P.citrinum

% Inhibition Concentrations Dry Mycelial SN Treatment or (mg/ml) wt. (gm) % Stimulation (-)

3 2.075 6.36 5 2.087 5.82 Azadiracta 1 10 2.112 4.69 Indica 20 2.088 5.78 50 2.256 -1.81 3 2.116 4.51 5 2.114 4.60 Eucalyptus 2 Globusa 10 2.016 9.03 20 2.015 8.53 50 2.027 9.07 3 2.111 4.74 5 2.054 7.31 Datura 3 10 2.067 6.72 Alba 20 2.088 5.78 50 2.123 4.20 3 2.081 4.42 5 2.025 4.92 Swietenia 4 10 2.107 4.51 Mahagoni 20 2.116 6.09 50 2.118 8.62 3 2.182 1.53 5 2.167 2.21 Cajanus 5 10 2.175 1.85 Cajan 20 2.194 0.99 50 2.262 -2.08 3 2.196 0.90 5 2.167 2.21 Andographis 10 2.135 3.66 6 peniculata 20 2.116 4.51 50 2.102 5.14 3 2.114 4.60 5 2.116 4.51 Silybum 7 10 2.106 4.96 Marinum 20 2.094 5.51 50 2.105 5.01

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3 2.109 4.83 5 2.097 5.37 Ocimum 8 10 2.103 5.10 Sactum 20 2.108 4.87 50 2.114 4.60 3 2.109 4.83 5 2.105 5.01 Mentha 9 10 2.112 4.69 piperta 20 2.098 5.31 50 2.112 4.69 3 2.107 4.92 5 2.079 6.18 10 Azolla 10 2.104 5.05 20 2.107 4.92 50 2.114 4.60 11 Control 2.216 00.00

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REFERENCES . Mossini, S.A.G., De, O.K.P. and Kemmelmeier, C. (2004). Inhibition of patulin production by P. expansum cultured with neem (Azadirachta indica) leaf extracts. Journal of Basic and Applied Microbiology. 44: 106–113. . Trease, G.E. and Evans M.C. (1983). Textbook of Pharmacognosy, 12th ed. Balliere Tindall, London. 343–383. . Tiwari, P., Kumar, K., Panik R., Pandey, A., Pandey, A. and Sahu P.K. (2011). Antimicrobial activity evaluation of the root of Carica papaya Linn. International Journal of PharmTech Research. 3(3): 1641-1648. . Shrivastava, J and Atri, D.C. (1998). Effect of homoeopathic drugs on the production

of aflatoxin B1 by Aspergillus flavus. Journal of Phytopathological Research. 11(1): 45-49. . Simone, A. G. Mossini, Carla, C. Arrotéia and Carlos Kemmelmeier (2009). Effect of Neem Leaf Extract and Neem Oil on Penicillium Growth, Sporulation, Morphology and Ochratoxin A Production. Toxins. 1: 3-13. . Parekh, J., Jadeja, D. and Chanda S. (2005). Efficacy of aqueous and methanol extracts of some medicinal plants for potential antibacterial activity. Turkish Journal of Biology. 29: 203-210. . Joshi, B., Sah, G.P., Basnet, B.B., Bhatt, M.R., Sharma, D., Subedi, K., Pandey, J. and Malla, R. (2011). Phytochemical extraction and antimicrobial properties of different medicinal plants: Ocimum sanctum (Tulsi), Eugenia caryophyllata (Clove), Achyranthes bidentata (Datiwan) and Azadirachta indica (Neem). Journal of Microbiology and Antimicrobials. 3 (1): 1-7. . Lee, S.O., Choi, G.J., Jang, K.S. and Kim, J.C. (2007). Antifungal activity of five plant essential oils as fumigant against postharvest and soilborne plant pathogenic fungi. Plant Pathology Journal. 23(2): 97-102. . Mahesh, B. and Satish, S. (2008). Antimicrobial activity of some important medicinal plant against plant and human pathogens. World Journal of Agriculture Science. 4(S): 839-843. . Mann, A., Banso, A. and Clifford, L.C. (2008). An antifungal property of crude plant extracts from Anogeissus leiocarpus and Terminalia avicennioides. Tanzania Journal of Health Research. 10(1): 34-38.

* Corresponding author Email - [email protected] Mobile no - +917697466446

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 45 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Evaluation of different media and grain substrates for mycelial growth of Pleurotus sajor caju

Poonam Dehariya*, Anjuli Choubey, and Deepak Vyas Lab of Microbial technology and Plant Pathology Department of Botany, Dr. H. S. Gour, University, Sagar (M.P.) E-mail:[email protected]

ABSTRACT Four culture media viz. Potato dextrose agar ( PDA), Malt extract agar (MEA), Czapeck's agar (CZ), Wheat extract agar (WEA) and three grains viz. wheat, maize and sorghum were analyzed for mycelial growth of Pleurotus sajor caju. Higher growth rate 4.33 cm/day was obtained on PDA whereas poor growth rate 2.99 cm/day was obtained on WEA media. Among the grains, used for spawn preparation, wheat grains are found best. Introduction-Pleurotus spp. commonly known as oyster fungus, is a commonly primary decomposer of wood and vegetal residues (Zedrazil and Kurtman,1981). The oyster mushrooms belong to the genus Pleurotus. They have a high saprophyte colonizing ability and can grow on virtually any agricultural waste. They rank among the top six mushrooms produced in the world. (Gibriel et al, 1996). Appreciated because of its delicious teste, this fungus has high quantities of proteins, carbohydrates, minerals (calcium, phosphorus, iron)and vitamins (thiamine, riboflavin and niacin) as well as low fat (Sturion and Oetterer,1995;Justo et al.,1998;Manzi et al.,1999).The production of this mushroom is dependent on the relation-ship between nutrient status of the substrates, strain and the prevailing environmental conditions (Lee et al,2007).To culture microbes in laboratory, suitable gradients are used for the preparation of substrate which they can used as food and these are called culture media. In present studies different culture media viz. Potato dextrose agar ( PDA), Malt extract agar (MEA), Czapeck's agar (CZ), Wheat extract agar (WEA) were used for mycelial growth of P. sajor caju. Many investigators have exerted their efforts to cultivate this mushroom on solid artificial media rather than submerged culture (Ryu et al, 1998). Similarly spawn is the seed material from which the artificial culture of any fleshy fungi can be attempted. It consists of mycelium and supporting medium. Sinden, 1973 stated that vegetative phase of a fungus is called mycelium and the material used to plant the bed is called spawn. In present study wheat, maize and sorghum grains were used for the spawn of P. sajor caju. Material and methods: The experiments were conducted in the lab of microbial technology and plant pathology, Department of Botany, Dr. H. S. Gour University,

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 46 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Sagar. Pure culture of P. sajor caju. was carried out in the JNKVV, Jabalpur. Isoloates were maintained by monthly transfers to potato dextrose agar medium (PDA) and stored in refrigerator after growth. Preparation of different media: One litre of PDA medium contained 200 gm. potato starch, 20 gm. dextrose, 20 gm agar-agar and 1000 ml distilled water. One litre MEA medium contained 20 gm. malt extract, 20 gm. dextrose, 20 gm. agar-agar,1000 ml water. One litre CZ agar medium contained 2 gm Sodium chloride, 1gm dipotassium phosphate, 0.5 gm potassium chloride, 0.01gm ferrous sulphate, 30 gm sucrose, 15 gm agar-agar, 1000 ml distilled water. One liter WEA medium contained 32 gm wheat grain, 20 gm agar-agar, 1000 ml distilled water. All media were sterilized in an autoclave at 15 Ib at 1210C for 30 min. and then poured in 90 mm Petri dishes under the laminar air flow to avoid contamination. Media were cooled to 400C. Fresh mushrooms were inoculated on culture media then cultures were incubated at 25 ± 10C for 7 days. Radial growth of mycelium in cm of different media was observed until the Petri dishes were filled with it. The experiment was repeated for 3 times. Preparation of spawn: For making spawn method of Mehta and Kumar, 1988 was followed. Clean whole grains are taken for the purpose. Broken grain should be avoided. The grains are pre-wetted by boiling in water for 20-30 min After boiling, excess water is drained off by spreading the grains on a wire mesh. Grains are now mixed with gypsum (Calcium sulphate) and Chalk powder (Calcium carbonate) at the rate of 2% and 0.5% respectively on dry weight basis (Garcha et al, 1981). The grains are now filled in containers and mouth of containers plugged with non-absorbent cotton. These are then sterilized in an autoclave at 22 Ib pressure for 1.5- 2 hours. This gives a uniform temperature of 126.50C, which is sufficient to kill bacterial and other contaminants, which might spoil the cultures afterwards. The grains are now allowed to cool in room temperature over night. Next day bottles are inoculated with two bits of agar medium cognized with the mycelium of pure cultures by putting the culture bits just opposite to each other in the inner side of glass surface in the middle of the bottle. About 7-10 days after inoculation, bottles are shaken vigorously so that mycelial threads are broken and mixed with grains. Three weeks after incubation, the stock culture becomes ready for further multiplication of spawn. Inoculated bottles are incubated at 26 ± 20C. Results and discussion: Media is defined as any substrate or material that will enable micro-organisms to grow and multiply. All media should provide carbon, nitrogen sources in addition to minerals and other growth factors for the growth of micro- organisms. Agar is a complex carbohydrate derived from algae of the genus

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 47 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 gellidium, this solidify the media. Present results reveals that PDA was the best tested media for fungal growth. Average rate of growth was 4.33cm./day. This medium is suitable for fungal (P. sajor caju.) growth. The second best medium was MEA. Rate of fungal growth was also high on CZ. Comparatively WEA media shows less mycelial growth of P. sajor caju. In this respect, Santiago (1983) found that mycelial development was marginally better on a medium containing glucose and sucrose than other carbon sources. The mycelial growth variation of six sp. of Pleurotus at different temperatures was studied by Mehta and Bhandal (1988). Asghar et al, (2007) were used PDA and MEA media for the growth of P. sajor caju. Gibriel et al, 1996 also used different media and observed growth rate of P. sajor caju mycelia. Kim et al, 2002 reported mycelial growth by submerged culture of various edible mushrooms under different media. Kumar et al, 2009 reported effect of growth parameters on mycelial culture and fructification of Pleurotus eryngii. Present results also reveals that wheat grains was best for mycelial growth of P. sajor caju followed by sorghum and maize. Jandaik and Kapoor, 1974 successfully produced the spawn of Pleurotus species on cereals, millets and other wastes such as saw dust and pearl millet. Effect of different spawn substrates on yield of Pleurotus sp. was studied by various workers (Jain and Vyas 2005; Garcha, 1981; Sivprakasam and Kandaswamy, 1981). Suman (1990) outlined the details of spawn preparations and characteristics of good spawn.. Gibriel et al, 1996 were used different organic substrates for the preparation of spawn of P. sajor caju. Asghar et al, (2007) also used wheat, sorghum and oat grains for the spawn of P. sajor caju. The goal of this work was to find out the good mycelia and spawn substrate for mycelial growth of P. sajor caju.

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 48 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Observation: Table;1 Comparison of different media for mycelial growth at 25±10C

Days after Media inoculation PDA MEA CZ WEA 1 0.466 0.3 0.2 0.23 2 1.8 1.63 1.5 1.4 3 2.76 2.66 2.5 2.2 4 3.63 3.0 3.5 2.8 5 5.5 4.46 4.2 3.5 6 7.63 5.83 5.5 4.8 7 8.53 7.33 6.5 6.0 R 4.33 3.60 3.41 2.99 CD(0.05) 0.146 0.28 0.166 0.36 Mycelial growth is given in mean. R = average of mycelial growth rate (cm/day).

Table:2 Mycelial running period in wheat, maize and sorghum grains.

Spawn-run days of spawn run S.N. (%) Wheat Maize Sorghum 1 25 3.33 5 5.33

2 50 6.66 8 8.33

3 75 10 11 12

4 100 15.33 18 19

1.33 2.06 1.68 CD (0.05)

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References:  Asghar, R. Tariq, M. and Rehman, T. 2007 Propagation of Pleurotus sajor caju (oyster mushroom) through tissue culture. Pak. J. Bot.,39 (4):1383-1386.

 Garcha, H. S., Sekhon, A., and Phutela, R.P. 1981 Utilization of agri-wastes for mushroom cultivation in India. Mushroom Science., 11, 1:245-254.

 Gibriel, A.Y., Ahmed, M., Rasmy, N., Rizk, I. and Abdel- Rehem, N.S.1996 Cultivation of oyster mushrooms (Pleurotus spp.):Evaluations of different media and organic substrates. Mushroom Biology and Mushroom Products, Royse (ed.) Penn. State University, 415-421.

 Jain, A. K. 2005. Thesis on Mushroom Cultivation with special reference to Pleurotus florida and their Marketing potential in Sagar Region.

 Jandaik C.L., Kapoor J.N. 1974; Studies on cultivation of Pleurotus sajor-caju (Fr.) Singer. Mush Sci. 9(1):667–672.  Justo, M.B., Guzman, G.A., Mejia, E.G., Diaz, C.L., Martinez, G. and Corona, E.B.1998.Composition quimica de tres cepas mexicanas de setas (Pleurotus ostreatus ). Archivos Latinomericanos de Nutricion 49, 1, 81-85.

 Kim, S.W., Hwang, H.J., Park, J.P., Cho, Y.J., Song, C.H., Yun, J.W.2002 Mycelial growth and exo-polymer production by submerged culture of various edible mushrooms under different media. Letters in Applied Microbiology,34,56-61.

 Kumar, A., Thakore, B.B.L. and Sharma, 2009. Effect of growth parameters on mycelial culture and fructification of Pleurotus eryngii. J. Mycol. Pl. Pathol.,39,1,19-22.

 Lee, YL, Huang GW, Liang ZC, Mau JL (2007). Antioxidant properties of three extracts from Pleurotus citrinopileatus. Food Sci. Technol. 40: 823-833.

 Manzi, P., Gambelli, L.,Marconi, S., Vivanti, V. and Pizzoferrato, L.,1999 Nutrients in edible mushrooms: An inter-species comparative study. Food Chemistry 65,4,477-482.

 Mehta, K.B. and Bhandal, M.S.1988 Mycelial growth variation of six Pleurotus species at different temperatures. Indian J. Mush., 14:64-65.

 Mehta, K.B. and Kumar, S. 1988. Studies on spawn production of white button mushroom Agaricus bisporus. Indian J. Mycol. Pl. Pathol.,18:269-271.

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 Ryu, Y. H., Yoon, Y. S., Jo, W.S., Park, S. D., Choi, B.S. and Kim, J.K. 1998 Effect liquid spawn on Flammulina velutipes cultivation. Korean Journal of Mycology 26,20-24.

 Sinden, J.W. 1973. Mushroom Experiments. Penn. Agr. Exper. Stn. Bull.352.

 Sivaprakasam, K.; Kandaswamy, T. K.. Mathan, K.K 1982. Effect of cultivation methods on sporophore production of Pleurotus sajor-caju (Fr.) Singer (Mushroom, yield) Agric J. 69 (10) 681-683.

 Suman, B.C. and Sharma, V.P. 1990 Screening of different varieties of wheat (Triticum aestivum Linn) for spawn production of Agaricus bisporus (Lange) Sing. in Himachal Pradesh. Indian J. Mycol. Pl. Pathol. 20, 1, 44-46.

 Sturion, G.L. and Otterrer, M.,1995.Composicao quimica de cogumelos comestiveis (Pleurotus spp.) originados de cultivos em differentes substatos. Ciencia e Tecnologia de Alimentos 15,2,189-193

 Sreesakthi, T.R. and Ponmurugan, P. 2006 Studies on the yield performance of oyster mushrooms with different biowastes. Paper presented in National Seminar on Emerging trends in industrial Biotechnology, Vivekanathan college of Engineering for women, Tiruchengode, Tamilnadu. Feb. 17 &18.

 Zadrazil, F. and Kurtzman, R.H., 1981. The biology of Pleurotus cultivation in the tropics In: Chang, S.T. and Quimio, T.H., Editors, 1981. Tropical mushrooms, The Chinese University Press, Shatin, Hong Kong, p. 493.

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Some Additions to the Coelomycetes from Central India

P. Chauksay, A.N. Rai, Naveen Verma and S.N. Tripathi Department of Botany. Dr. H.S. Gaur University, Sagar 470003 Madhya Pradesh, India Email- [email protected]

Abstract During frequent surveys for phytoparasitic foliicolous microfungi, two interesting specimens were collected from Sagar Forest Circle of Madhya Pradesh, which upon detailed examination proved to be undescribed taxa of Microsphaeropasis liliacearum sp. nov. and Phloeospora paramaculans sp. nov. infecting Ruscus aculeatus Linn. (Liliaceae) and Ficus benghalensis Linn. (Moraceae) respectively. These are illustrated, described and compared with closely related species in the genera. Microsphaeropasis liliacearum Chauksay, Rai, Verma and Tripathi sp. nov. (Fig.1) Maculae amphigenosae, circulares vel irregulares, peraeutns delimitatus. Coloniae amphyllosae ad maculae limitatae, interdem expando folii, atro. Mycelium hypharum immersum, ramosum, septatum, hyalina vel pallide brunneae. Conidiomata pycnidial, globosa vel subglobosa, discreta, immersum vel superficiale, fusce brunneae vel atro, unilocularis, constitutus parietibus tenuibus brunneae cellulae, usque 182 µm dimetro. Ostiolata singularis, circularis, centralis. Conidiophori absentia, conidiogenosae cellulae enteroblasticae, phialidicae, determinatae, discretae, ampulliformes, leavia, hyalina, 5-8 x 6-8 µm. Conidia globosa vel subglobosa vel oblonga, simplicia, laevibus parietibus, aseptata, plerique fuscis parietibus, pallide brunneae 3-12.5 x 2.5-11 µm. Lesions amphigenous, circular to irregular, usually towards the leaf margin in apical region of leaf, sharply delimited from the healthy tissue by a choloritic halo. Colonies amphyllous, confined to the spots, sometimes spreading over the leaf, represented by very fine dots, black. Mycelium of hyphae immersed branched, septate, hyaline to light brown. Conidiomata pycnidial, globose to subglobose, separate, immersed to superficial, dark brown to black, unilocular, composed of thin walled brown cells, up to 182 µm in diam. Ostiole single, circular, central, conidiophores absent. Conidiogenous cells enteroblastic phialidic, determinate, discrete, ampulliform, smooth, hyaline, 5-6 x 6-8 µm. conidia spherical to subspherical to oblong, simple, smooth walled, aseptate, usually thick walled, light brown, 3-12.5 x 2.5-11 µm. (Fig. 1)

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Hab. on leaves of Ruscus aculeatus Linn. (Liliaceae), Octobe, 1988, University Botanical Garden, Sager, leg. M. Gupta, RG 53, type, IMI 333067. A thorough study and survey of literature of different species of Microsphaeropsis on various hosts (Sutton, 1971, 1974, 1980 a & b, Morgan-Jones, 1975) shows that present collection has been found to exhibit some similarities with M. cellista(H. Syd.) Sutton, M. globulosa (Camera) Sutton, (Sutton, 1980a) and M. clidemiae Sutton (Sutton, 1980b), as in Table 1.

A critical look to the Table 1 reveals that the authors’ collection is related to M. callista, M. globulosa and M. clidemiae, resemblance being only in conidial structure. At the same time it differs in dimensions of conidia, conidiomata and conidiogenous cells. Moreover, symtomatology of M. clidemiae also differs from proposed species (epigenous against amphigenous). Therefore, it is justified to describe it as a new species. Phloeospora paramaculans Chauksay, Rai, Verma and Tripathi sp. nov. (Fig. 2) Maculae amphigenosae, circulares vel irregulares, dispersae per totam superficiem folii, albidus atro. Coloniae amphiphyllosae, ad maculae limitatae, stapitis praeditae atro. Mycelium hypharum, immersum, ramosum, septatum, hyalina. Conidiomata acervular, subepidermalis, tenuibus parietibus sclerotioides formatus. pallide vel medio divacea, 50-225 µm. dimetro, conidiophori absentia. Conidiogenosae cellulae holoblasticae, discretae, indeterminatae, annellidecae, annellationibus formatus de supra cellulae acervulares, parvae cylindrata, laevia, subhylina vel pallide olivaceous, 4-17 x 1-1.5 µm. Conidia simplicia, solitaria leavia, septati, usque 1-4 transversae septati, eguttulata, cylindrata vel falcate, parum curvata, gradasim acuminate apices, apices acuta et obtusa vel subtruncuta bases, hyalina, 15-48 x 1-2 µm. Lesions amphigenous, circular to irregular, distributed all over the leaf surface, whitish black. Colonies amphiphyllous, confined to the spot, represented by very fine dots, black. Mycelium of hyphae immersed, branched, septate, hyaline. Conidiomata acervular, subepidermal, thin walled textura angularis. Light to mid olivaceous, 50-225 µm in diam. Conidiophores absent. Conidiogenous cells holoblastic, discrete, indeterminate annellidic, annellations formed from the upper cells of acervuli, short cylindric smooth, subhyline to light olivaceous, 4-17 x 1.15 µm. conidia simple, solitary, smooth, septate, upto 1-4 transversely septate, eguttulate, cylindric to falcate, slightly curved, gradually tapered towards the apex, apex acute and obtuse to subtruncate base, hyaline, 15-48 x 1-2 µm. Hab. on living leaves of Ficus benghalensis Linn. (Moraceae), October, 1980, Naryawali, North Sagar Forest Division, leg. M. Gupta. RG 15, type IMI 329134.

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Of all the species of Phloeospora described so far, P. Maculans (Phloeospora state of Mycospharella mori) is the only earlier described species found comparable with the authors’ collection in Table 2, reported on Moraceae. A critical llok to the Table reveals that the proposed taxon resembles P. maculans (Bereng.) Allesch. (Sutton, 1980) in structure of conidiomata and conidial colour and septation. However, P. parmaculans is quite dissimilar in diameter of conidiomata, length of conidiogenous cells structure of conidia and conidial dimensions as against P. maculans. These points of divergence justify that these two cannot be conspecific. Therefore, this collection merits description and illustration as a new species. Acknowledgements The authors are grateful to the Director, International Mycological Institute, Kew, (U.K.) for the accession of the deposited fungal specimens and the Head, Department of Botany, Dr. H.S. Gour University, Sagar for providing facilities. The financial assistance from UGC Research Award Scheme to ANR is thankfully acknowledged. References Sutton, B.C. (1971). Coelomycete-IV. The Harknessia and similar fungi Eucalyptus. Mycol. Pap. 123 : 1-46. Sutton, B.C. (1971). Miscellaneous Coelomycete on Eucalyptus. Nova Hedwigia. 25 : 161-172. Sutton, B.C. (1980a). The Coelomycete. Fungi Imperfecti with Pycnidia, Acervuli and Stromata. C.M.I., Kew, England. Sutton BC. 1980b. Microsphaeropsis clidemiae sp. nov., associated with leaf lesions on Clidemia hirta. Trans Br Mycol Soc 74 (3) : 645-647. Morgan, Jones G. (1975). Concerning some species of Microsphaeropsis. Can. J. Bot. 52 : 2575-2579.

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Fig. 1: Microsphaeropasis liliacearum sp. nov. A. Symptom B. Conidiomata C. Conidiogenous cells D. Conidia

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Fig. 2: Phloeospora paramaculans sp. nov. A. Symptom B. Conidiomata C. Conidiogenous cells D. Conidia

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Two new species of the genus Leptoxyphium Speg. from the forest flora of Madhya Pradesh, India

S. Shrivastava, A.N. Rai and Naveen Verma Department of Botany Dr. H.S. Gaur University, Sagar 470003 Madhya Pradesh, India Email- [email protected] Abstract During frequent surveys for foliicolous microfungi, two interesting specimens were collected from Sagar Forest Circle of Madhya Pradesh, after detailed mycotaxonomic treatment they found to be new taxa of Leptoxyphium Speg. Viz., L. hemidesmiicola sp. nov. and L. tamarindus-indicae sp. nov. infecting Hemidesmus sp. (Linn.) R. (Periplocaceae) and Tamarindus indicus Linn. (Caesalpiniaceae) respectively. These are described, illustrated and compared allied taxa. Leptoxyphium hemidesmiicola Shrivastava, Rai and Verma sp. nov. (Fig.1) Maculae amphigenosae, micro ad irregulares plurimum reperio juxta the vertex vel margo de the folii laminae, griseus. Coloniae epiphyllosae, effusae, atra, in acervus de pulvis gleba. The hypharum es coalitus de plures vel minus cylindrata cellulae they es formae an irregulares reticulatus opus. Synnemata orientes de cellula hyphis co 6 vel 10 hypharum comprehendo a normalae crescoae synnemata et quia maximus de eorum diuturnitas. The synnamata hypharum es non ramosum et the cellulae es longior dein crassus, formae a globosus, terminales conidiogenosae zona. They es plures vel minus cylindrata, plures conflatus ad wardae basis dare the espectus de pycnidium. et partim. The synnamata mensuram 66-332 X 33-66µm hyalina. Phialoconidia aggregata in limosus globulus ad apex de synnemata. they es fere micro globosa vel subglobosa, eseptatus, interdum conidia similis apparentes ex catenata, subhyalina vel brunneae 8-16 X 8-16µm. Lesions amphigenous, small to irregular mostly found at the tip or margin of the leaf lamina, grey. Colonies epiphyllous, effuse, black, in cluster of powdery mass. The hyphae are composed of more or less cylindrical cells. They are forming an irregular network. Synnemata arise from a short celled hyphae cemented together, upto 6 to 10 hyphae comprise a normal developing synnemata. The synnamata hyphae are unbranched and the cells are longer than broad, forming a globular, terminal, conidiogenous zone. They are more or less cylindrical, occasionally more swollen towards the base giving the appearance of pycnidia, hyaline. The synnamata measures 66-332 X 33-66µm. in diam. Phialoconidia gather in a slimy globule at the apex of synnamata. They are usually small globose to sub globose, aseptate, some conidia are found in chain, sub hyaline to brown, 8-16 X 8-16µm.

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On living leaves of Hemidesmus sp. (Linn.) R. (Periplocaceae). Oct. 2000, Pathariya Jat, Sagar, Madhya Pradesh, India, leg. S. Shrivastava S.U. Herb No. SR- 110 holotype, HCIO 44, 227. L. fumago (Woronichin) Shrivastava 1982 and L. graminum (Patouillard) Speg. (Hughes, 1976) are the two reported species taken for comparison with the described fungal (Table-10). The data of the table reveal that the author's fungus is only a little bit comparable and that too only in general characters while the same is at great variance in the characters of mycotaxonomic importance such as structure and dimensions of synnemata and conidia including the symptomatology. Looking to the condition it seems proper for time being to keep this as a separate species of Leptoxyphium. Leptoxyphium tamarindus-indicae sp. nov. Shrivastava, Rai and Verma sp. nov. (Fig.2) Maculae indistintus. Coloniae amphyphyllosae, fere reperio in apicalis regio de the folium, effusae, floccosus, obscure atra. Synnemata hyphaerum coalitus de plures vel minus cylindricus cella ad septum constrictae, irregulares network ramosum each synnematous cella multus plures produco and amplus. Synnemata arising apo the gleba de repent hyphae, each hyphae aceus 3-6 polytreticis ex unitus conjunctim formae synnema they are uselly conflatus istorsum basis vultus destromae synnemata mensuram 49-140 X 6-18.5µm obscure brunneae vel atra. Phialoconidia aggregata im limosus globulus ad apex de synnemata postquam igenus realesed frome the mucosus massa they are unicelluler globosa vel. subglobosa, interdum conidia similis apparentes ex catenata, glabrae, exilis leptodermus eseptatus, hyalina vel brunneae 3.12-6.24 X 2-6µm. Lesions indistinct. Colonies amphiphyllous, mostly found on the apical region of the leaf, effuse, cottony, dark black. Synnematous, hyphae, composed of more or less cylindrical cells constricted at the septa forming irregular network, branched, each synnamatous cell much more elongated and broad. Synnamata arising from the mass of repent hyphae. Each hypha made of 3-6 rows. Each hyphae join together forming synnamata. They are usually swollen towards the base giving the appearance of stroma. The synnamata measures 49-140 X 6-18.5µm. dark brown to black.. Phialoconidia aggregated in slimy globule at the tip of synnamata, after bearing realesed form the slime mass they are unicellular, globose to sub globose, some conidia are giving appearance of catenate, smooth thin walled, aseptate, hyaline to brown 3.12-6.24 X 2-6. µm. On living leaves of Tamarindus indicus Linn. (Caesalpiniaceae). Oct. 2000 University Campas Sagar (M.P.) India, leg. S. Shrivastave S.U. Herb No. SSR 74 holotype HCIO No. 44,226.

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L. fumago (Woronichin) Shrivastava 1982, is the species taken into comparison with the proposed species (Table-12). If we look to the tabular data and morphotaxonomic characters of the two, it becomes clear that the author's collection is quite distinct and can not be accommodated but for a little bit of similarities. Therefore, the situation demands for its inclusion as a new species. Acknowledgements The authors are grateful to The Curator, Herbarium Cryptogamiae Indiae Orientalis (Indian Agricultural Research Institute) New Delhi for depositing the fungal specimens and their accession and the Head, Department of Botany, Dr HS Gour University, Sagar for providing necessary facilities. The financial assistance received through UGC Research Award Scheme to ANR is also gratefully acknowledged. References Shrivastava, R.C. 1982. Leptoxyphium fumago (Woron.) R.C. Shrivastava, Arch. Protistenk, 125(1-4):333. Hughes, S.J. (1976). Sootymolds, Mycologia, 2: 693-821.

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Figure 1 : Leptoxyphium hemidesmiicola sp. nov. A. Symptom, B. Synnema, C. Phialoconidia

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 64 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Figure 2 : Leptoxyphium tamarindus-indicae sp. nov. A. Symptom, B. Synnema, C. Phialoconidia

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Ganoderma basal stem rot of tropical trees of Patharia forest of Sagar (M.P) Javed Ahmad Wagay, Imtiyaz Ahmad Sheikh, Poonam Dehariya and Deepak Vyas Lab of microbial technology and Plant pathology Department of Botany, Dr. H.S.Gour Central University, Sagar (M.P.) Email: ufaq07@ rediffmail.com /[email protected]

ABSTRACT Ganoderma stem rot is caused by the fungus Ganoderma sp. The fungus degrades or rots the lower 4-5 feet of the trunk. A number of trees are hosts of this fungus. . Symptoms may include wilting (mild to severe) or a general decline. The disease is confirmed by observing the basidiocarp (conk) on the trunk. This is a hard, shelf- like structure which is attached to the lower 4-5 feet of the trunk. However, not all diseased trunks produce conks prior to death..A plant cannot be diagnosed with Ganoderma basal rot until the basidiocarp (conk) forms on the trunk or the internal rotting of the trunk is observed after the plant is cut down. The fungus is spread by spores, which are produced and released from the basidiocarp (conk).Conditions that are conducive for disease development are unknown.There are currently no cultural or chemical controls for preventing the disease or for curing the disease once the plant is infected. A plant should be removed as soon as possible after the conks appear on the trunk. Eradicate as much of the stump and root system as possible of the infected plant, because the fungus survives in the soil for longer time. Planting another plant back in the same location where the infected plant has been already removed is not recommended. INTRODUCTION Ganoderma is a basidiomycetes,lamillales fungus belonging to Polyporaceae family. The genus was created by Karsten in 1881 based on Polyporus lucidus Leys:Fr.All Ganoderma species lack cystidia, have echinulate basidiospores and cause white rot in their substrata. Knowledge of species of Ganoderma Karsten (Ganodermataceae) has been, and still is rather chaotic, principally due to their polymorphism(Ryvarden,1991:2000).The taxonomic criteria are diverse considering different authers(Gottlieb and Wright,1999),so the correct name for many taxa used in different works remained unclear (Moncalvo and Ryvarden,1997).Ganoderma species employ wood like resource as saprotrophs as well as parasites as it produces certain enzymes which degrade the soft tissues and effect the xylem thus causing serious threat to transduction of water and minerals of the plant. In nature it grows in densely wooded mountains of high humidity and dim lighting.It flourishes mainly on the

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 66 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 dried trunks of dead aged trees.Out of 10,000 aged dead trees approximately 2% will have Ganoderma growth,therefore it is very scarce indeed (Willard,T.,1990) . Present study was conducted at Sagar and its surrounding area . Sagar is situated near the north of Tropics of Cancer on Vindhyan peaks with 230-50″ North latitude and 780- 40″ East longitude. The forest is a dry deciduous forest having an undulating topography with low rising hills scattered all round. The average monthly minimum and maximum temperatures are 11.1°C and 25°C in January and 40°C and 44°C in June. Forest is dominated by Tectona grandis,, Butea monosprma, Accacia sp.,Santalum album,Delbergia sisso,Delnix regia etc. and ground flora consists of Albezia procera.,Bambosa sp.,Lantana camara, Parthenium hysterophorus, Euphorbia geniculata, E.hirta, Heteropogon contortus, Cyanadon dactylon, Bryophyllum sensitivum, Cassia tora etc. The study reveals that the most of the dominant trees species especially Mimmopsis elangi, Santalum album, Albizia procera , Delbergia sisso and Delnix regia of the forest are being victimized by a disease known as Ganoderma basal white rot which is caused by species of Ganoderma namely, G.lucidium,G.aplantum and G.tsuga.The disease is more prevalent in the University campus and in farm houses, situated down in the valley , where Mimmopsis elengi, Butea monosperma and Santalum album are in bulk abundance. Pathogen and Hosts The fungal genus Ganoderma is a group of wood-decaying fungi which are found throughout the world on all types of wood-gymnosperms, woody dicots and palms. There are many different species of this fungus in Sagar but only the pathogen G.lucidum was found wide spread among the trees. Other species of Ganoderma like G. zonatum which was reported to occur mostly in palms but also reported its occurrence on non palm hosts, However these reports are very limited. Therefore, palms are considered the primary hosts of G. zonatum (Lonsdale,1999). Te basidiocarp (conk) on a tree was observed, especially when it is still living, it is safe to assume it as G. lucidum and not some other Ganoderma species. G. lucidum species are often observed on hardwoody trees such as Dalbergia sisoo, Santalum album and Delnix regia and rarely found on other living plants. The other Ganoderma species may occur on dead trunks and stumps, but they act simply as saprobes .A number of plants are assumed to be susceptible to this disease. While not all trees in University campus of Sagar have been documented with Ganoderma basal stem rot. Those not documented with this disease may be considered escaped. The only possible exception of such plant species will be to have some natural antagonistic which do not allow fungus to grow.

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Table-1 : The table shows Occurance of different Ganoderma species on different hosts in different sites of University campus.

Host Pathogen Site/Location Mimmopsis elengi Ganoderma aplantum Road side,University campus Mimmopsis elengi Ganodeerma tsuga Deptt. of Botany Delbergia sisso Ganoderma lucidium Deptt. of Law Carica papaya Ganoderma lucidium Residential quater, University campus Delnix regia Ganoderma lucidium Botanical garden,university campus. Santallum alba Ganoderma lucidium English deptt.,university campus. Dead trunk Ganoderma sp. Deptt.of Law,University campus. Tamarindus indica G.tsuga Convent school,Sagar Butea monosperma Ganoderma lucidium Boys hostel,University campus Murraya koenigii Ganoderma lucidium Farm house.Patheria Accasia sp. G.tsuga Botanical garden (aboratum.) Terminalia arjuna Ganoderma sp. Garphera (Sagar) Dead trunk Ganoderma lucidium Botanical garden,university campus. Dead trunk Ganoderma sp. University ground

Symptoms, Signs and Diagnosis G. lucidum is a white rot fungus which produces numerous enzymes to degrade (rot) woody tissues primarily lignin and cellulose. As the fungus destroys wood internally, the xylem (water conducting tissue) is eventually effected. Therefore, the primary symptom of the Ganoderma basal rot disease which can be observed is wilting (mild to severe) of all leaves out of spear leaf(fig-1). Other symptoms can best be described as a general decline- slower growth and off –color foliage.

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Fig-1.Mimmopsis elengi shows wilted and dry leaves due to Ganoderma applanatum infection The basidiocarp or conk is the most easily identifiable structure associated with the fungus. The conk originates from fungal growth inside the tree trunk. Figure- 2 illustrates different stages in the development of the conk. When the conk first starts to form on the side of a trunk or stump, It is a solid white mass which is relatively soft when touched. It has an irregular to circular shape and is relatively flat on the trunk or stump. As the conk matures, a small shelf or bracket will start to form as the basidiocarp begins to extend or protrude from the trunk. It is still be white, both on the top and bottom surfaces. Eventually, it forms a very distinct shelf –like structure that is quite hard with a glazed reddish-brown top surface and a white undersurface. A mature conk have a half moon shape with relatively “straight” side directly attached to the trunk. If a conk is present on the trunk at the same time, the wilt or decline symptoms will appear, then it is safe to diagnose Ganoderma basal rot. However, It is not common for conks to appear prior to severe decline and death of a tree, In that situation, the only way to determine Ganoderma basal rot is to cut cross-section through the lower 4 feet of the trunk soon after the tree has been cut down.

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 69 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Fig.2.Three phases of Basidiocarp (Conk) development of G.lucidium, ‘white button” top of picture is the begning stage of conk. The lower left structure is a mature conk and the lower right structure is old conk.

Fig.3.Basidiocarp (conk) of Ganoderma Sp. Neither “Stem” nor “stalk” was found which attaches the conk to the trunk

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 70 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Conk of G. zonatum can be upto 8 inches at their widest point and 2 inches thick. However, conks take on the shape and size of the area in which they are growing. Microscopic basidiospores are produced in the “pores” present on the underside of the conk. When a masss of basidiospores are present on a white surface,they appear brownish-red in color. Objects immediately around a conk which has dropped its spores may appear to be covered with rusty colored dust. One conk can produce at least 3 cups of spores at a time. Disease and Fungus life cycle The fungus spreads primarily by the spores produced in the basidiocarp (conk).The spores become incorporated into the soil, germinate and the hyphae (fungal threads) then grow over the tree roots. The fungus does not rot the roots, it simply uses the roots as a means of medium to move to the woody trunk tissues. Once a tree is infected with Ganoderma, the fungus moves surrounding locations in which other plants are growing and reaches to the soil associated with other trees resulting its fast spreading infection.

Fig.4. Cut stump with numerous basidiocarps (conks) of G. sp. It is unknown to us exactly how many months or years pass between initial infection of trees and development of the conk. There is no particular method till today to determine and declare Ganoderma infection of a tree before the appearance of conk. Therefore, it is not possible guarantee that plants are free of Ganoderma when first planted in the landscape (Schwarze et al ,2000).

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Figures 3 and 4 are examples of the wood rotting and disease progression patterns observed. The fungus colonizes and degrades the tree trunk tissue closest to the soil line first, expands in diameter at the base and moves up the center or near- center of the trunk. Therefore, the disease progression pattern within the trunk is best described as cone-shaped, widest at the soil line and narrowing to a pinpoint.

Fig-4. Spores releasing from mature conks resulted in reddish brown appearance of conks and surrounding area Disease Management No environmental conditions or landscape management practices have been observed which suppress the development of Ganoderma basal stem rot. The disease occurs in natural settings and is highly maintained. It occurs on woody trees which have been maintained very well nutritionally (no nutrient deficiencies). The fungus killed trees which have no apparent mechanical injuries and those which have been severely damaged by weed trimmers. Soil type appears to have no relationship with disease either, as diseased plants have been observed on limestone rocks. There has been no discernible pattern to provide clues as to why some trees become infected and die from Ganoderma and others do not. In general, the fungus is being located in the lower 4-5 feet of trunk and has three implications. First, fungus does not spread with pruning tools because it is not associated with leaves. Second, the lower portion of the trunk could be chipped and used for mulch in the landscape,third, only the lower 4-5 feet of trunk needs to be protected from the fungus. However, typical xylem- limited and systemic fungicides are not effective unless they are capable of spreading beyond the vascular tissue and protecting all the wood in the lower portion of the trunk. No fungicide can be effective once the conks have formed, since a large

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 72 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 percentage of the trunk cross-sectional area has already been destroyed . Since we have no means of predicting or determining which tree is infected with Ganoderma. This effectively eliminates the use of fungicides as a control method, either preventively or curatively for the present time. Therefore, there are no fungicide recommendations for this disease. Since basidiospores from the basidiocarps (conks) are probably the primary method of spreading the fungus, trees should be monitored closely, especially after a tree has died or been removed for any reason. The fungus will readily colonize and degrade tree stumps. Once the fungus becomes established in this dead wood, it will normally produce conks with millions of basidiospores which are easily spread by wind and water (Flood et al ,2000).Therefore, it is essential to monitor trees and their stumps for the conks, remove the conks and place in trash receptacle that will be incinerated or delivered to a land fill. The early removal of conk reduces spore release. It was important to observe that trees shoud be regularly monitored because if any symptom of disease appears,it should be taken care of immediately.Since spores blow with the wind, so it should be a community effort to reduce the spread of the spores of this lethal fungus. Once a conk is observed on a tree, the tree should be removed primarily for safety reasons. This is especially important during the season. As indicated before, if conks are being produced on a live tree, it means that a significant portion of the trunk is already rotted. These trees are likely to be the first blown down in heavy winds. Remove the complete plant including rotten portion. Any plant material left behind will be a host for the fungus as its spores are prominently survives in the soil. It has been observed that replaced tree planted into the same site where a tree died from Ganoderma basal stem rot also became diseased and died. Therefore, replanting with another tree is risky. Other plant species (wood trees, shrubs, etc.) are also affected by Ganoderma sp. (Elliott and Broschat,2006; Mohd Suud et al, 2007). If one replanting a tree, one must then follow the guidelines properly. Remove the stump and all roots from the site and fumigate the soil by a licensed professional legally registered fumigant for the landscape. An example would be the product of dazomet (trade name = Basamid). If a tree is located at a site surrounded by concrete material, remove all of the old soil, bring in new soil and then fumigate. However, this does not guarantee the new tree will remain free of Ganoderma basal stem rot, as the fungus may already be associated with the new tree and fungal spores can be easily blown into the newly fumigated site.

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References  Elliot, M.L. and Broschat, T.K. (2001). Observation and Pathogenecity experiments on Ganoderma zonatum in Florida. Palms. 45 : 62-72.

 Elliot, M.L.and Broschat, T.K.(2006). Ganoderma butt rot of Palms. Institute of Food and Agricultural Sciences, University of Florida, Gainesville.17:213- 218.

 Flood , J.,Bridge,P.D. and Holderness,M.(2000).Ganoderma diseases of perennial crops.CABI publishing,UK,275-279.

 Flood, J.,P.D. Bridge and Holderness, M. eds. (2000). Ganoderma diseases of perennial crops. CABI publishing, Wallingford, U.K.21:23-29.

 Gottlieb,A.M.,Wright,J.E.(1999).Taxonomy of Ganoderma from South America:sub genus Ganoderma.Mycological Research.Cambridge.103:661- 673.

 Moncalvo,J.M.,Ryvarden,L.(1997).Anomenclatural study of the Ganodermat aceae.Synopsis Fungorum .Fungiflora.11:114.

 Ryvarden,L.(1991).Genera of Polypores nomenclature and taxonomy.Syno psis Fungorum. Fungiflora.5:363.

 Ryvarden,L.(2000).Studies in neotropical polypore. A preliminary key to neotropical species of Ganoderma with a lacate pileus.Mycolgia.92:180-191.

 Lonsdale,D(1999). Principles of tree hazard assessment and managemet. Plant pathology. 42:321-328 .

 Schwarze,F.W.(2000).Developement of pro-progression of decay in the sap wood of living trees.Arboricultural journal .25:321 -337.

 Idris,A.S, Arifin, D.,Swinburne,T.R. ,Watt,T.A. (2000).The identity of Ganoderma species responsible for basal stem disease of palm oil in Malaysia-morphological characteristis.MPOB information Series ,TT No:77.

 Willard,T. (1990).The Reishi Mushroom: Herb of Spiritual potency and Medical Wonder.Syivan Press .23: 4-7.

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Distribution of VA-mycorrhizal fungi in different wheat cultivars in Sagar Region O.P. Dwivedi1 and D. Vyas2 1- P.G. Department of Botany, S.V.M. Science & Tech. P.G. College, 2- Lalganj, Pratapgarh, (U.P.) INDIA 2- Department of Botany, Dr. H. S. Gour University Sagar, (M.P.) INDIA

ABSTRACT Thirteen different wheat cultivars planted in 1M2 micro plots at departmental botanical garden, Sagar M.P. were examined for the VAM infection. All the wheat cultivars were found to be infected with vesicular arbuscular mycorrhizae. However, their population in rhizosphere and root infection varied to a considerable extent from species to species. Present study indicates the frequent occurrence of Acaulosporaceae with dominating of Acaulospora species and rare occurrence of Entrophospora species, in addition to members of Glomaceae and Gigasporaceae. However, members of Glomaceae recorded the highest species diversity at all the test sites. Glomus mosseae (LMSS), Glomus fasciculatum (LFSC) and Glomus hoi (LHOI) were found as most frequent (above 70%) VAM fungal species in all the four study sites while, Glomus ambisporum (LABS) with above 70% frequency occurred in almost all the sites excepting site IV Keywords: VA-mycorrhizal fungi, wheat cultivar The distribution, abundance and life history of many plants and animals can easily be observed without any specialized or sophisticated techniques. In addition the taxonomy and classification of many plant and animal groups is well established to the species level. This information form a sound basis from which it is easier to pursue experimental studies of their ecology, evolution and role within natural ecosystem. In the case of some organisms, particularly those below ground, the difficulty of the task of discussing their taxonomy, distribution, abundance and life history hinders meaningful experimental investigations of their role in natural ecosystem. One such group of organisms is the zygomycete fungi of the order Glomales, which are commonly known as VAM fungi. The VAM fungi form the Vesicular –arbuscular mycorrhizal symbiosis with the roots of plants and in many cases this association is beneficial to both partners (Harley and Smith, 1983). The Glomalean fungi associated with the majority of the land plant species are found worldwide in virtually all habitats. Arbuscular mycorrhizas are ubiquitous with 90% of terrestrial plant species forming the AM symbiosis. They form large multinucleate spores in the soil, but growth of hyphal stage is dependent on the association with plant roots. Taxonomists have described three families, six genera and about 150

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 75 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 species, most of which are in the genus Glomus. This taxonomy is based on morphological characters and especially on features of the spores. A single morphospecies e.g. Glomus mosseae may however, has a very wide distribution both ecologically and geographically. The study of VAM fungi has been impeded by their obligate biotrophic nature and by difficulties in the identification of spores, especially in the field material. In this situation, molecular techniques have enormous potential and there have been a number of important resent advances. Both mitochondrial DNA and the multicopy nuclear ribosomal RNA (r-RNA) gene have been used for species identification and phylogenetic analysis in fungi (Bruns et al. 1991). In mycorrhizal symbiosis formed between plant roots and the arbuscular mycorrhizal (AM) fungi, the Glomales is of great interest to botanists because of its potential influence on ecosystem processes, its role in determining plant diversity in natural communities and the ability of these fungi to induce a wide variety of growth responses in coexisting plant species. Little attention, however, has been paid to the ecological role of diversity of AM fungi. AM fungi have been shown to play a significant role in the floristic diversity and structure of annual and perennial plant communities. However, in most of these ecological investigations, little or no attention has been paid to diversity of AM fungi themselves. This is because experiments conducted in pots have indicated that AM fungi show little host specificity. Consequently it was thought that few selection pressures that it favors extensive divergence arise in mutualistic symbiosis. The lack of basic information about the AM fungi stems from a few fundamental difficulties encountered in their identification, culture and taxonomy, coupled with the great difficulty in manipulating AM fungi in natural ecosystem without greatly modifying their environment in other ways. Some of the problems of identification and possibly of taxonomy can be using various techniques. In this chapter we outline briefly as to why the study of AM fungal diversity warrants greater attention. Understanding the real, rather than the potential significance of AM fungal diversity in natural communities has posed the greater problem. For a given plant community, we need to know how diverse the AM fungal community is, which plant roots are colonized by which AM fungi, whether, there is seasonality in the pattern of colonization, whether any specificity between plant and AM fungi occurs and what the effect of those AM fungi will be on plant and the ecosystem. However, the difficulty of identifying AM fungi in the roots of plants has always been an obstacle to their study in natural communities though the identification of AM fungi in roots based on morphological observation has been successful in pot experiment, the

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 76 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 usefulness of this technique for field investigations appears to be limited because the hyphal morphology of one AM fungal isolate is likely to vary with host species. Our interest in diversity in VA mycorrhizas arose from considerations of differences in mycorrhizal efficiency i.e. the variation in nutritional benefits and costs to the plant that are associated with differences in amounts of inorganic nutrients such as P or Zn transferred to the plant in return to organic carbon (e.g. Sugar) transferred to the fungus. Previously, it can be widely assumed that bi-directional transfer of inorganic nutrients and organic carbon occurs solely across arbuscules. Indeed in their taxonomic revision of the Glomales, Morton and Benny (1990) emphasized on this assumedly unique role of the arbuscules as a fundamental feature of the order. Diversification of AM fungi The AM symbiosis is ancient; the fossil records suggest that it occurred in the first land plants. Morphological diversification is low, with only 152 recognized species in six genera (Walker and Trappe, 1993).Law (1985) pointed out that considering the age of the symbiosis this diversification is low, and he suggested that this is typical of mutualistic symbiosis, as there are few external pressures which would favor the selection of new traits. However, the low divergence of AM fungi seems at odds with their functional diversity. Information regarding the genetic diversity of AM fungi can also be obtained using molecular techniques and the results of such investigations can and have helped to construct a phylogenetic classification of AM fungi (Simon et.al. 1993). Until recently, methods for studying AM fungal diversity and the ecology of the symbiosis have been wholly reliant on the morphology of the spore phase. Spore of AM fungi are relatively large, easy to extract from soil and have several morphological characters that allow species identification to be determined by experienced personnel. However, the use of spore data alone for the assessment of ecosystem diversity and ecology has long been recognized as unsatisfactory. The relationship between the morphological diversity of AM fungi and their genetic and functional diversity has not been established. According to Walker (1992) many described taxa are “workable” in that they are recognizable and are found repeatedly in different part of the world, albeit in diverse environments. For example, several species of AM fungi which were reported in a semi-arid site in Australia (Mc Gee, 1989) also occurred in a species rich meadow in the north of England (Sanderes, 1993), both ecosystem comprising completely different soils, vegetation and climate. This indicates that AM fungi are very plastic in their environmental adaptability. Therefore the present study was undertaken to find out the biodiversity of VAM fungi, which is naturally occurred in the rhizosphere soils of wheat at four different study sites of this region.

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MATERIALS AND METHODS The present study represents an attempt to establish the qualitative and quantitative distribution of VAM fungal species in rhizosphere soils of wheat. The study was taken up in four different sites eg. I - Sagar, II- Khurai, III- Banda and IV- Deori of Sagar region (M.P.) during November 1999 to October 2001. Isolation and Identification of VAM Spores The rhizosphere soil samples from 10-20 cm depths were collected from 13 cultivars i.e. HI-1502, HI-1500, GW-1172, HD-2781, HW-2004, MACS-3208, MPO-1126, A-9-30-1, HI-1501, HD-4694, HD-4672, C-306 and Sujata of wheat plants. The soil samples were collected then processed by wet sieving and decanting techniques of Gerdemann and Nicolson (1963) to isolate VAM spores. Spores were flushed in to petridishes and counted under a steriozoom dissecting microscope. Only healthy spores were counted. Spores were mounted in polyvinyl alcohol- lactoglycerol (PVLG), examined for their various morphological characters and identified with the help of key provided by Schenck and Perez (1987),Mehrotra (1997), Mehrotra and Baijal (1994), Hall and Fish (1979) and species code of VAM fungi were followed after Perez and Schenck (1990). Frequency Distribution of VAM fungi Frequency of different species of VAM fungi was calculated at four different test sites on the basis of their occurrence with selected wheat cultivars.

RESULTS Identification and Morphological Description of VAM species About 160 species (Dalpe, 1997) of VAM fungi have been isolated and identified from different geographical areas or habitats. In the present study, 82 VAM species were isolated and identified from the rhizosphere soil of 13 different wheat cultivars from four different study sites of Sagar region. The fundamental problem in studies of the distribution of VAM fungi lies in identifying the fungi, because the VAM fungi can neither, be cultured in the absence of living root nor isolated on agar plates by standard microbiological techniques. The taxonomy of VAM, which form VAM, has not been completely clarified. It relies almost entirely on spore morphology, which may change with spore age. Another difficulty in determining the distribution of VAM fungi is that spores of all species are easy to recover from soil, because they are either very small or are non randomly dispersed in dense sporocarps of small spores.

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The spore collected from pure lines of VAM fungi, maintained on certain host plant and cultivated in soil under sterile conditions. In present study both qualitative and quantitative morphological characters have been considered for description and identification of VAM fungi. The possible identification was done up to species level. Frequency and Diversity of VAM fungi Population of VAM fungi from four different study sites represented 82 different species in the rhizosphere soils of 13 selected wheat cultivars and these belongs to six genera viz. Acaulospora, Entrophospora, Gigaspora, Glomus, Sclerocystis and Scutellospora. Out of these, 77 VAM fungal species were identified up to species level and the remaining 5 species could be identified only up to genus level. These five new species were identified on the basis of their different morphological features. The genus Glomus was most dominant VAM fungi constituted up to 62.2% of the total isolates followed by Acaulospora (21.9%) then Scutellospora (6.1%), Sclerocystis (4.9%), Gigaspora (3.7%) and least was Entrophospora (1.2%). LMSS, LFSC and LHOI were found as most frequent (above 70%) VAM fungal species in all the four study sites while, LABS with above 70% frequency occurred in almost all the sites excepting site IV (Fig. 1).

Table 1: Diversity of VAM fungi from total (82) isolated spp. in rhizosphere soils of wheat cultivars at different test sites

Frequency of VAMF Occurrence of VAMF (%) Study from total From sites (82) total Glomus Acaulospora Scutellospora Sclerocystis Gigaspora Entrophospora isolated isolated spp. spp. spp. spp. spp. spp. species spp.

I 80 97.60 61.25 22.50 6.25 5.00 3.73 1.25

II 73 89.02 60.30 24.70 5.40 4.10 4.10 1.40

III 78 95.12 60.26 23.07 6.41 5.13 3.85 1.28

IV 75 91.46 60.00 22.70 6.70 5.30 4.00 1.30

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In site I, 80 species (97.6%) occurred out of total isolates (82 species) of VAM fungi. The highest percentage of VAM fungi occurred in genus Glomus (61.25%) followed by Acaulospora (22.5%) then Scutellospora (6.25%), Sclerocystis (5.0%), Gigaspora (3.75%) and lowest percentage occurred in Entrophospora (1.25%) (Table 1, Fig. 1.1). The most frequent VAM fungal species (above 70% frequency) occurred with rhizosphere soils of different selected wheat cultivar was LMSS (92.3%); LFSC,

LHOI (84.61%); LABS, Glomus Sagarai (PSN) and CPLC (76.92%), while LPBS (7.69%) was least frequent at the site I (Table 2). In site II, out of 82 isolates, 73 species of VAM fungi (89.2%) were recorded with the rhizosphere soils of selected wheat cultivar. The highest percentage of VAM fungi occurred in Glomus (60.3%) followed by Acaulospora (24.7%) then Scutellospora (5.4%), Gigaspora, Sclerocystis (4.10%), and lowest in Entrophospora (1.4%) (Table 1, Fig. 1.2). Result shows that the most frequent VAM fungal species (above 70% frequency) was LFSC (92.3%), LABS (84.61%); GABD, LGSP, LHOI, LMSS AND CCLS (76.92%). However, LFLV (7.69%) showed least frequency with the rhizosphere soils of selected wheat cultivar (Table 2). In site III, 78 species (95.12%) occurred out of 82 isolates of VAM fungi; in which the highest percent of VAM fungal species occurred in Glomus (60.26%) followed by Acaulospora (23.07%) then Scutellospora (6.41%), Sclerocystis (5.13%), Gigaspora (3.85%) and least was observed with Entrophospora (1.28%) (Table 1, Fig. 1.3). The most frequent VAM fungal species (above 70% frequency) were recorded as LFSC (90.9%), LMSS (81.81%), GABD, LGSP and LHOI (72.72%), while LIVM, LSTL and SPKS (9.09%) showed little frequency with all the selected wheat cultivars (Table 2).

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Among all the sites, site IV had 75 (91.16%) different VAM fungal species. The highest VAM fungal species occurred with Glomus (60.0%) followed by Acaulospora (22.7%) then Scutellospora (6.7%), Sclerocystis (5.3%), Gigaspora (4.0%) and lowest percent of species was recorded with Entrophospora (1.3%) (Table 1, Fig. 1.4). In this site the most frequent VAM fungal species (above 70% frequency) were recorded as LFSC (81.81%), Acaulospora omeae (PSN), LCRD, LHOI, LMSS,

Glomus sagarai (PSN) and CMNT (72.72%). The VAM fungal species GRSA, LGBF and SPKS (9.01%) showed poor frequency with all the selected wheat cultivars (Table 2). DISCUSSION The VAM fungi belong to a very old category of Zygomycetes and have been recently regrouped in a single order, the Glomales (Morton and Benny, 1990), which include all species capable of living in symbiosis with plant. The bulk of known species belong to the family Glomaceae, which include the genera Glomus and Sclerocystis. The arbuscular mycorrhizal fungi consist actually of approximately 160 species belonging to three family and six genera and have a worldwide distribution. The bulk of known species have been described over the last two decades, which indicates the increased interest in these organisms and also the difficulty inherent in their taxonomic treatment. In fact, the greatest difficulty is that the entire taxonomy of these organisms is currently based on the morphological characters of the spores. They are single-celled structures, of generally globoid shape, with thick walls made up of several layers of different textures, connected to the filamentous network by a suspensor hypha of varied morphology. Since the morphological characters are reduced and often variable depending on the maturity of the spores studied, ultrastructural studies serve to support observations made previously by optic microscopy. Arbuscular mycorrhizal (AM) fungi are ubiquitous in the terrestrial habitats invading over 80% of the land plants. The structural and functional aspects of this association, as evident from fossil records, appear to be quite concerned through time (Harley and Smith, 1983). This benevolent relationship has not only helped the emergence of first land plants but also supported further successional establishment in widely diversified environments, viz. agricultural soils, mine soils, coal wastes, alkaline soils, desert soils and other habitats. The colonization of VAM fungi varies greatly with various factors such as soil fertility, soil type, species of mycorrhizal fungi and plant cultivars. Before the significance of Mycorrhizal fungi play a significant role in growth and yield of plant (Dwivedi, 2004). The generic diversity of AM fungi in four sites of Sagar region studied here appears to be more elaborated than those reported from any other part or any other

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 81 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 types of soils, where species are restricted to Glomaceae and Gigasporaceae, with species in Glomaceae dominating over others. In contrast, this study indicates the frequent occurrence of Acaulosporaceae with dominating of Acaulospora species and rare occurrence of Entrophospora species, in addition to members of Glomaceae and Gigasporaceae. However, members of Glomaceae recorded the highest species diversity at all the test sites. CONCLUSION As such on the basis of results of present study it may be concluded that AM fungi are not host (different cultivars of wheat) specific rather they formed symbiotic association with any of the cultivars grown here. Though any change in environmental conditions directly influence the morphology of VAM fungi resulting appearance of new species. Five new species of VAM fungi belonging to the genus Glomus and Acaulospora (viz. Acaulospora omeae, Glomus cardiseptum G. dentatae, G. sagarai and G. vyaseae) associated with the wheat crop of Sagar region are being reported for the first time.

REFERENCES 1. Bruns, T.D., White, T.J. and Taylor, J.W. 1991. Fungal molecules systematic. Anu. Review Ecol. Systematics. 22 : 525-564.

2. Dalpe, Y. 1997. Biodiversity of mycorrhizal fungi. Report for the third meeting of the subsidiary Body on scientific, technical and technological advice (SBSTTA), convention on Biodiversity held at Montreal Quebec Canada on September 1 to 5, 1997: 1-13.

3. Dwivedi, O.P. 2004. Effect of VA-Mycorrhiza on Winter Wheat Genotype C- 306. J. of Mycol. and Pt. Pathol. 34 (2): 623-625.

4. Gerdemann, J.W. and Nicolson, T.N. 1963. Spores of mycorrhizal Endogone sp. extracted from soil by wet sieving and decanting. Trans. Br. Mycol. Soc. 46: 235-244.

5. Harley, J.L. and Smith, S.E. 1983. Mycorrhizal symbiosis. Academic Press. London. New York: 483.

6. Hall, I.R. and Fish, B.J. 1979. A Key to the Endogonaceae. Trans. Br. Mycol. Soc. 73. 261-270.

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7. Law, R. 1985. Evolution in a mutualistic environment. In: The biology of mutualism: Ecology and evolution. (ed.) Boucher D.H. London: Croon Helm: 145-170.

8. Mc Gee, P.A. 1989. Variation in propagule number of vesicular – arbuscular mycorrhizal fungi in a semi-arid soil. Mycological Res. 92: 28-33.

9. Mehrotra, V.S. 1997. Problem associated with morphological taxonomy of AM fungi. Mycorrhiza News. 9: 1-10.

10. Mehrotra, V.S. and Baijal, U. 1994.Advances in the taxonomy of vesicular- arbuscular mycorrhizal fungi. Biotechnology in India : 227-286.

11. Morton, J.B. and Benny, G.L. 1990. Revised classification of arbuscular mycorrhizal fungi (Zygomycetes): New orders, Glomales, tow new suborders, Glomineae and Gigasporineae and tow new families, Acaulosporaceae and Gigasporaceae, with an emendation of Glomaceae. Mycotaxon, 37: 471-491.

12. Perez, Y. and Schenck, N.C. 1990. A Unique code for each species of VA- mycorrhizal fungi. Mycologia. 82. 256-260.

13. Sanderes, I.R. 1993.Temporal infectivity and specificity of vesicular – arbuscular mycorrhizas in co-existing grassland species. Oecologia. 93: 389- 355.

14. Schenck, N.C. and Perez, Y. 1987. Manual for identification of VAM fungi. Synergistic Pub. Gainesville. Fl., U.S.A.

15. Simon, L., Bousquet, J., Levesque, C. and Lalone, M. 1993. Origin and diversification of endomycorrhizal fungi and coincidence with vascular land plants. Nature. 363: 67-69.

16. Walker, C. 1992. Systemics and taxonomy of the arbuscular endomycorrhizal fungi (Glomales)- a possible way forward. Agronomic. 12: 887-897.

17. Walker, C. and Trappe, J.M. 1993. Names and epithets in the Glomales and Endogonales. Mycological Res. 97: 339-344.

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Table 2: Frequency distribution of VAM fungal species in rhizosphere soils of selected wheat cultivars at different study sites Frequency VAM fungal (%) S. No. species Study Sites I II III IV 1. ABRT 61.53 53.84 54.54 63.63 2. ADLC 69.23 46.15 36.36 27.27 3. ADTC 30.76 30.76 45.45 45.45 4. ADLT 53.84 46.15 45.45 36.36 5. AFVT 23.07 38.46 27.27 18.18 6. AGDM 61.53 69.23 63.63 63.63 7. ALCN 69.23 61.53 54.54 36.36 8. ALVS 53.84 46.15 63.63 54.54 9. AMLL 61.53 54.15 63.63 63.63 10. AMRW 30.76 30.76 27.27 - 11. AMYC 23.07 23.07 18.18 18.18 12. ANCS 38.46 38.46 27.27 36.36 13. A. omeae(PSN) 61.53 61.53 54.54 72.72 14. ARHM 46.15 53.84 45.45 36.36 15. ASCB 69.23 61.53 63.63 45.45 16. ASPN 53.84 30.76 27.27 18.18 17. ASPC 46.15 38.46 36.36 36.36 18. ATRP 46.15 38.46 45.45 45.45 19. EIFQ 30.76 46.15 36.36 45.45 20. GABD 69.23 76.92 72.72 63.63 21. GMRG 23.07 30.76 27.27 45.45 22. GRSA 30.76 23.07 18.18 9.09 23. LAGR 53.84 61.53 45.45 54.54 24. LABD 23.07 38.46 54.54 54.54 25. LABS 76.92 84.61 72.72 63.63 26. LAST 23.07 15.38 - 36.36 27. LBTR 30.76 38.46 36.36 45.45 G. 28. 46.15 53.84 63.63 45.45 cardiseptum(PSN) 29. LCTC 46.15 53.84 54.54 63.63 30. LCRD 69.23 61.53 54.54 72.72 31. LCLR 38.46 38.46 27.27 18.18 32. LDLH 23.07 23.07 18.18 27.27 33. G. dentatae(PSN) 38.46 46.15 63.63 54.54 34. LDST 46.15 46.15 45.45 36.36 35. LDPH 30.76 30.76 36.36 27.27 36. LDMR 46.15 38.46 63.63 63.63 37. LETC 30.76 46.15 45.45 36.36 38. LFSC 48.61 92.30 90.90 81.81 39. LFCS 69.23 61.53 45.45 45.45

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40. LFLS - 46.15 45.45 45.45 41. LFRG 15.38 - 27.27 - 42. LFLV 23.07 7.69 18.18 - 43. LGSP 84.61 76.92 72.72 54.54 44. LGDM 46.15 38.46 36.36 63.63 45. LGBF 15.38 23.07 - 9.09 46. LHTS 69.23 53.84 54.54 45.45 47. LHOI 84.61 76.92 72.72 72.72 48. LINR 30.76 15.38 - 27.27 49. LIVM 15.38 23.07 9.09 - 50. LLCT 23.07 - 18.18 27.27 51. LLPT 38.46 30.76 45.45 45.45 52. LMCC 61.53 46.15 54.54 54.54 53. LMCL 53.84 53.84 63.63 45.45 54. LMGC 38.46 38.46 27.27 27.27 55. LMLS 30.76 30.76 36.36 36.36 56. LMNS 23.07 - 27.27 45.45 57. LMSS 92.30 76.92 81.81 72.72 58. LMTC 38.46 46.15 45.45 45.45 59. LMST - 38.46 36.36 - 60. LOCT 23.07 30.76 36.36 - 61. LPLD 23.07 - 27.27 45.45 62. LPSH 7.69 - 36.36 18.18 63. LPBS 69.23 38.46 - 27.27 64. LPVN 15.38 30.76 54.54 54.54 65. LPST 23.07 38.46 54.54 63.63 66. LRDT 30.76 23.07 27.27 - 67. LRTC 23.07 15.38 18.18 18.18 68. G. sagarai(PSN) 76.92 61.53 54.54 72.72 69. LSTL 23.07 - 9.09 18.18 70. LSGT 30.76 23.07 54.54 63.63 71. LTRT 23.07 15.38 27.27 45.45 72. LVSF 46.15 - 36.36 45.45 73. G. vyaseae(PSN) 53.84 61.53 54.54 63.63 74. SCCG 61.53 46.15 54.54 54.54 75. SPCC 61.53 46.15 63.63 63.63 76. SPKS 23.07 - 9.09 9.09 77. SRBF 38.46 38.46 27.27 27.27 78. CCLS 61.53 76.92 63.63 54.54 79. CFLG 38.46 46.15 54.54 45.45 80. CMNT 69.23 53.84 63.63 72.72 81. CPLC 76.92 46.15 45.45 45.45 82. CWRS 69.23 - 36.36 54.54 (- Absent, PSN- Proposed species name)

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Glomeromycota of medicinal plants:Abundance and community structure influenced by the soil depth Deepak Vyas Lab of Microbial Technology & Plant Pathology Dr. H.S. Gour University Sagar (M.P.)

Abstract The present study deals with the diversity and distribution of VAMF at different sites with different selected plants. Maximum number of VAMF species were found at site IV (57 species) out of which Glomus species was most dominant (58%), followed by Acaulospora (19%), Scutellospora (8%), Sclerocystis (4.8%) and Gigaspora (1.6%) respectively. In site II 56 species of VAMF were observed with Glomus (55%), followed by Acaulospora (22.5%), Scutellospora (8%), Gigaspora (1.6%) and Sclerocystis (3.2%) respectively. In site III 55 species of VAMF occurred with Glomus (51.6%) followed by Acaulospora (22.5%), Scutellospora (9.7%), Sclerocystis (4.8%) and Gigaspora (0%) respectively. In site I 54 species of VAMF were found; out of these Glomus was highest 53% followed by Acaulospora (22.5%), Scutellospora (5%), Sclerocystis (1.6%) and Gigaspora (1.6%) respectively. These results suggest that selected study sites are rich in VAMF frequency and diversity.The Shanon-Wiever index confirms that diversity of VAMF fungal species varies with the test plant and maximum diversity was observed with Ocimum sanctum (3.948), and Withania somnifera (3.909) respectively. Maximum ANOVA value recorded in case of and Withania somnifera (0.20) and Ocimum sanctum (0.19) respectively. Maximum richness value was observed in case of Ocimum sanctum (0.3948) and Withania somnifera (0.0391). Introduction Mycorrhizae is the mutualistic symbiosis (non-pathogenic association) between soil borne with the roots of higher plants (Quilambe., 2003), revealed that they are found in wide range of habitats usually in the roots of angiosperms, gymnosperms and pteridophytes.They also occurs in the gametophytes of some mosses, lycopods and psilotes, which are rootless (Mosse et al., 1981; Vyas et al., 2007, 2008). AMF have shown to be potentially able to take up both organic (Hodge, Campbell & Fitter, 2001) and inorganic nitrogen from the soil (Govindarajulu et al., 2005). VAM fungi are essential components of ecosystem for both re-vegetation of the degraded lands and maintenance of soil structure (Caravaca et al.,2005), therby reducing the risks of erosion and desertification.

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Soil characteristics, plant species, and climate may all regulate the arbuscular mycorrhizal (AM) fungi community. The distribution of certain VAM fungal species has been related to soil pH, phosphorus level, salinity, soil disturbance (Abbott and Robson 1991), vegetation (Johnson et al., 1992), or hydrologic condition of the soil (Ingham and Wilson, 1999; Miller and Bever, 1999). In general terms, increases in soil pH, nutrient status and salinity in soil are related to a decrease in VAM root colonisation or spore density (Abbott and Robson, 1991). Despite the importance of VAM fungi in the physiology and nutrition of plants, as well as in shaping plant communities (Grime et al., 1987; Van der Heijden et al., 1998; Smith et al,. 1999), factors affecting the presence, diversity, spore density, and root colonisation by AM fungi in soil are poorly understood. One reason is the difficulty of establishing causation from correlation of soil and plant factors with VAM fungal populations. Another reason is that AM fungi can associate with a wide range of hosts present in community, but the sporulation rates of AM fungi have been found to be host dependent (Bever et al., 1996; Lugo and Cabello, 2002). Host-dependence of VAM fungal population growth rates in soil may play an important role in the maintenance of VAM fungal species diversity in grasslands (Bever et al., 1996), and suppression of mycorrhizal symbioses may result in a decreasing of the dominant plant and an increase in species diversity (Hartnett and Wilson, 1999). In addition, plant diversity may increase or decrease if the dominant plant competitors are more weakly or more strongly mycotrophic than their neighbours (Hartnett and Wilson, 1999). An additional factor influencing populations of VAM fungi in soil, which may in turn affect the performance of plant species relative to each other, is the hydrologic condition of the soil, which may vary seasonally. The hydrologic condition of the soil plays an important role in determining plant community structure, and this effect is even more important when soils are commonly subjected to periods of dryness and flooding (Chaneton et al., 1998). VAM fungi have been found in the roots of many plants in wetlands (Ingham and Wilson, 1999; Miller and Bever (1999), or salt marshes (Brown and Bledsoe 1996). This is relevant because the fungi are believed to require wellaerated soils, and are thought to have problems adapting to flooded conditions (Mosse et al., 1981). Nevertheless, little is known of VAM fungi patterns in wetlands or of the influence of the hydrologic condition of the soil on populations of AM fungus species. The most important and historical account of medicine in the form of ‘Ayurveda’(2500 t0 900 B.C.), which is considered as ‘Upaveda’ ,Charak Samhita and Susruta Samhita’ also delt with plants related to medicine and their use in health management.These days many people cultivating medicinal plants to fulfil the increasing demands of pharmaceutical industries. The major biochemical constituents of Ashwagandha (is a small, woody shrub in the Solanaceae family) that root are

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 88 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 steroidal alkaloids and steroidal lactones in a class of constituents called withanolides. At present, 12 alkaloids, 35 withanolides, and several sitoindosides from this plant have been isolated and studied. A sitoindoside is a withanolide containing a glucose molecule at carbon 27. Much of ashwaganda's pharmacological activity has been attributed to two main withanolides, withaferin A and withanolide D. Tulsi, Queen of Herbs, the legendary "Incomparable One" of India, is one of the holiest and most cherished of the many healing and health-giving herbs of the Orient. Current research offers substantial evidence that Tulsi protects against and reduces stress; enhances stamina and endurance; increases the body's efficient use of oxygen; boosts the immune system; reduces inflammation; protects against radiation damage; lessens aging factors; supports the heart, lungs and liver; has antibiotic, antiviral and antifungal properties; enhances the efficacy of many other therapeutic treatments; and provides a rich supply of antioxidants and other nutrients. Thus prompted with above mentioned facts we undertook present study to understand how AM fungi play their role in association with test medicinal plants so that their bio-fertilizing potential can be exploited accordingly. Material & Method For the present investigation two test sites were selected, (I) Kariaya Village (II) Jaitpur Village. The experiments were conducted for quantitative and qualitative estimation of AM fungi were done from rhizosphere and non-rhizosphere soil and roots of test plants. The rhizosphere soil and root samples of selected test medicinal plants were collected from study sites up to 0-10, 10-20, 20-30, 30-40 cm depth. The VAM spores were isolated from the collected soil samples by wet sieving and decanting method (Gerdemann and Nicolson, 1963). Mycorrhizal spores are identified according to their spore morphology by conventional taxonomic key of Schenck and Perez (1990&http:/www.invam.cat.wu.edu). For the estimation of AM spores, a technique provided by Gour and Adholeya (1994) was followed. The soil pH was determined in 1:5 suspension of soil; deionized water ratio, electrometrically by glass electrode pH meter 335 (Jackson, 1982). Statistical analysis of data for comparison of means, analysis of variance (ANOVA), etc. was followed after Gupta and Kapoor (1997). Result The result obtained from the present study depicted in the table 1, which show the relative abundance of VAMF spores associated with test plants Withania somnifera and Ocimum sanctum, growing in the Karaiya village and Jaitpur village. Variance in relative abundance (RA) of VAMF spores along with the depths of the

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 89 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 soil was observed. Maximum value recorded of VAMF spores was observed from the soil surface up to 10 cm length and it reduces and minimum was recorded at 30-40 cm depth. Shannon-Weaver diversity index and evenness of VAM fungi is shown in table (2). The Shannon-Weaver index value suggests that W. somnifera harbours more diverse morphotypes of O. sanctum. Comparatively soil of Jaitpur village has fairly greater number of morphotypes in W. somnifera then in the soil of Karaiya, H 2.351 and H 2.250 respectively. However, Shannon –Weaver index (H') values confirm that (VAMF) reduces whereas in contrast H' value obtained from the different depth of rhizosphere of O. sanctum growing in Jaitpur village soil showed to maximum value at the depth of 10-20 cm (2.143), and further deeper region showed linear decrease an H` value. However, O. sanctum growing Karaiya village showed maximum H` value up to 10 cm depth and below this H` value gradually decreased The value obtained for evenness (J') of VAMF with W. somnifera growing in Karaiya or Jaitpur village soil are also described in table (2). Interestingly there is little hike in J` value at 20-30 cm and 30-40 cm deep in respectively with W. somnifera plants growing in Jaitpur village soil. However, same species growing at Karaiya village dose not show any significant difference in J` value. Evenness of VAMF J` value of O.sanctum in both the sites Karaiya village soil and Jaitpur village soil showed poorly different trend. At Kariaya village soil J` value almost remains same upto 30 cm depth, but suddenly significant reduction in J` value was observed. In contrast to this Jaitpur village soil though J` value remains same upto the depth of 30 cm but a significant increase in J` value at 40 cm depth was recorded. During the present study, a total of 27 morphologically distinct VAM species were isolated from the rhizosphere of Withania somnifera and Ocimum sanctum growing at two different sites; Karaiya village and Jaitpur village (Fig.1). Out of 27 VAM fungal species, 13 different species were found associated only with W. somnifera, six species were found only with O. sanctum and eight species were found common in both the plants. Thus, a total of 21 species associated with W. somnifera and 14 species were found associated with O. sanctum. Among the 21 VAM species found associated with W. somnifera, five VAMF species viz. Acaulospora mellea, A. scrobiculata, Glomus claroideum, G. etunicatum and G. macrocarpum were not found in Jaitpur soil, whereas A. bireticulata, A. denticulata, G. dimorphicum were not found in Jaitpur village soil (Fig. 2). Acaulospora sp., A. nicolsonii, G. clarum and G. hoi were the prominent species of the VAM fungi which were isolated from surface to 40 cm. depths in the Karaiya village soil. G. intraradices and G. mosseae were isolated from the depth of 30 cm. A. denticulata and Glomus sp. were obtained from the depths of 10-20 and 20-

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 90 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

30 cm. G. ambisporum, and G. fasciculatum were isolated from 0-10 and 10-20 cm depths. A. bireticulata, G. australe, G. desrticola, G. dimorphicum, and G. pustolatum were isolated from 0-10 cm depth in the Karaiya village soil (Table 1). In the Jaitpur village soil, A. nicolsonii, G. clarum, G. hoi and G. intraradices were isolated from the topsoil to of 40 cm depth. G. etunicatum, G. mosseae and G. versiforme were collected from of 30 cm depth. A. mellea and G. desrticola were isolated from 0-10, 10-20, and 30-40 cm soil depth. A. scrobiculata, G. australe, G. fasciculatum, G. macrocarpum, and G. pusotlatum were isolated from 0-10 and 10-20 cm depth. Acaulospora sp. and Glomus sp. were isolated from 0-10 and 20-30 cm depth. G. ambisporum was isolated only 10- 20 cm (Table 1). Out of 27 VAMF species, 14 species were found associated with O. sanctum in both the sites (Fig. 1). Among the 14 VAMF species, three species viz. A. foveata, Entrophospora infrequens and G. etunicatum were not found in Karaiya village soil (Fig. 2). A. nicolsonii and G. clarum were the two VAMF species found very prominent in Karaiya village soil and isolated in all measured soil depth. A. spinosa, G. fasciculatum, G. heterosporum and G. hoi were isolated from the depth of 30 cm. Whereas, A. scrobiculata, G. ambisporum and G. intraradices were isolated from the depth of 20 cm. G. botryoides was isolated in topsoil (0-10 cm) and Scutellospora pellucida was isolated from 20-30 and 30-40 cm soil depth (Table 1). In Jaitpur village soil G. clarum, G. fasciculatum and G. intraradices were isolated from 40 cm depth. A. nicolsonnii, G. heterosporum and G. hoi were collected from 30 cm depth while, A. foveata, G. ambisporum and G. etunicatum 20 cm depth. A. spinosa was isolated from 10-20, 20-30 and 30-40 cm soil depths, respectively. Here, also G. botryoides was isolated from the topsoil. E. infrequens was isolated from 20-30 and 30-40 cm depth and S. pellucida was isolated from 30-40 cm depth (Table 1). The result shown in fig.3 clearly indicate that 14 VAMF species associated with W. somnifera, commonly occurring in both the sites Karaiya village soil as well as Jaitpur village soil. Among 14 VAMF species, 11 species associated with O. sanctum. It was also observed that 6 VAMF species viz. A. nicolsonii, G. ambisporum, G. clarum, G. fasciculatum, G. hoi and G. intraradices were found associated with both the test plants at in both the sites. However, three species A. bireticulata, A. denticulata and G. desrticola which are associated with W. somnifera were found only in Karaiya village soil. A linear regression analysis with coefficient of determination (= squared correlation coefficient or r2) of VAMF spore population with soil depth, soil pH, and soil moisture per cent in W. somnifera and O. sanctum at both the sites were presented in fig. 4 (A-F) and fig. 5 (A-F). It is clearly evident from the result that the VAMF spore

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 91 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 population showed a strong negative correlation with soil depth, pH and moisture of the soil. It is assumed that an increase in single variable (depth pH, or moisture) resulted in decrease in VAMF spore population in both the test plants at both the sites. In Karaiya village soil, depth and moisture of rhizosphere soil of both the test plants show highly significant correlation, while, variation found in correlation between soil pH and spore population of both the plants. Here, VAMF spore population had weak correlation (r2= 0.563) with the pH of rhizosphere soil with W. somnifera in comparison to O. sanctum (r2 = 0.943). Whereas, in Jaitpur village soil, VAMF spore population showed similar trend as observed at Karaiya village soil with the depth and percent moisture of rhizosphere of both the plants. These two attributes significantly, correlated with the VAMF spore population (Fig. 5 A-F). The data presented in table (3) show the comparative analysis of average values of soil pH, soil moisture, VAMF spore population and Shannon-Weaver diversity index at four soil depths from both the sites. The mycorrhizal population dropped significantly from the upper to lower soil depth level. Both the soils showed similar relationships for depths and mean total spore population (Fig. 6). In the present study average soil moisture present initially increased two fold with the increasing depth (Fig. 7). Average soil pH found increased. Interestingly, soil pH values showed a general tendency to increase with increasing soil depth in both the site (Fig.8).

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 92 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 93 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 94 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 95 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Acaulospora bireticulata Acaulospora denticulata Acaulospora foveata Acaulospora mellea Acaulospora nicolsonii Acaulospora scrobiculata Acaulospora spinosa Archaeospora gerdemannii Entrophospora infrequens Glomus ambisporum Ocimum sanctum Ocimum Glomus australe Glomus botryoides Glomus claroideum Glomus clarum Glomus deserticola Withania somnifera Glomus dimorphicum Glomus etunicatum Glomus fasciculatum Glomus heterosporum Glomus hoi Glomus intraradices Glomus macrocarpum Glomus mosseae Glomus pustolatum Glomus versiforme Paraglomus occultum Scutellospora pellucida

Common

Fig. 1: Distribution of VAMF species in the rhizosphere soil of Withania somnifera and Ocimum sanctum

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 96 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

VAM Fungi

Karaiya Jaitpur Village Village

Acaulospora bireticulata

Withania Acaulospora denticulata Withania somnifera somnifera

Acaulospora foveata

Acaulospora mellea

Acaulospora scrobiculata

Entrophospora infrequens

Glomus claroideum

Glomus deserticola

Ocimum Ocimum sanctum Glomus etunicatum sanctum

Glomus macrocarpum

Fig. 2: Occurrence of VAMF species associated with either Withania somnifera or Ocimum sanctum growing in Karaiya village and Jaitpur village

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 97 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

VAM Fungi

Karaiya Jaitpur Village Village

Acaulospora nicolsonii

Acaulospora scrobiculata

Acaulospora spinosa

Archaeospora gerdemannii

Glomus ambisporum

Glomus australe

Glomus botryoides Ocimum sanctum Ocimum

Glomus clarum

Glomus dimorphicum

Glomus fasciculatum

Withania somnifera Glomus heterosporum

Glomus hoi

Glomus intraradices

Glomus mosseae

Glomus pustolatum

Glomus versiforme

Paraglomus occultum

Scutellospora pellucida

Karaiya Jaitpur village village

Fig. 3: Common occurrence of VAMF species associated with Withania and Ocimum either growing in Karaiya village or Jaitpur village

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40 y = -0.0433x + 39.094 40 y = -0.0517x + 39.388 r2 = 0.8876 r2 = 0.9255 35 35 30 30 25 25 20 20

15 15

10 10

5 Regressed Soil Depth (cm) 5 Regressed Soil Depth (cm) 0 0 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 AMF Spore Population VAMF Spore Population

(A) (D)

6.55 y = -0.0006x + 6.557 7.7 y = -0.0015x + 7.6659 2 2 6.5 r = 0.563 7.6 r = 0.9432 7.5 6.45 7.4 6.4 7.3 6.35 7.2 6.3 7.1 Regressed Soil pH Regressed Soil pH 6.25 7 6.2 6.9 6.15 6.8 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600

AMF Spore Population VAMF Spore Population

(B) (E)

1.8 1.8 1.6 1.6 1.4 y = -0.0016x + 1.7957 1.4 y = -0.0015x + 1.7101 2 2 1.2 r = 0.7825 1.2 r = 0.7701 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 Regressed Soil Moisture (%) Regressed Soil Moisture (%) 0 0 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 AMF Spore Population VAMF Spore Population

(C) (F)

Fig. 4 : Regression of VA mycorrhizal fungal spore population with soil depth; soil pH; soil moisture percent in Withania somnifera (A-C) and Ocimum sanctum (D-F) at Karaiya village

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35 40

30 35 30 25 25 20 20 15 y = -0.0338x + 36.032 15 y = -0.0503x + 41.974 2 2 10 r = 0.9989 r = 0.9106 10 5

Regressed Soil Depth (cm) Regressed Soil Depth (cm) 5 0 0 0 200 400 600 800 1000 0 100 200 300 400 500 600 700 VAMF Spore Population VAMF Spore Population

(A) (D)

6.45 y = -0.0006x + 6.4606 2 7.45 6.4 r = 0.7623 7.4 y = -0.0008x + 7.511 6.35 2 7.35 r = 0.8282 6.3 7.3 6.25 7.25 6.2 7.2 6.15 6.1 7.15 Regressed Soil pH 6.05 Regressed Soil pH 7.1 6 7.05 5.95 7 0 200 400 600 800 1000 0 100 200 300 400 500 600 700 VAMF Spore Population VAMF Spore Population

(B) (E)

2 y = -0.0014x + 1.999 y = -0.0017x + 2.0514 2 2 r2 = 0.9196 r = 0.9353 1.8 1.8 1.6 1.6 1.4 1.4 1.2 1.2 1 1 0.8 0.8 0.6 0.6 0.4 0.4

Regressed Soil Moisture (%) 0.2 Regressed Soil Moisture (%) 0.2 0 0 0 200 400 600 800 1000 0 100 200 300 400 500 600 700 VAMF Spore Population VAMF Spore Population

(C) (F)

Fig. 5 : Regression of VA mycorrhizal fungal spore population with soil depth; soil pH; soil moisture percent in Withania somnifera (A-C) and Ocimum sanctum (D-F) at Jaitpur village

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900 0-10 10-20 20-30 30-40 Soil Depth (cm) 800 700 600

500 Fig. 6: VA mycorrhizal spore 400 population per 100 gm of 300 rhizosphere soil of test medicinal 200 plants at two different sites in

VAMF Spore Population different soil depth 100 0 W. somnifera O. sanctum W. somnifera O. sanctum

Karaiya Village Jaitpur Village

2.5 0-10 10-20 20-30 30-40 Soil Depth (cm)

2

1.5

1 Soil Moisture (%) 0.5 Fig. 7: Soil moisture percent of rhizosphere soil of 0 test medicinal plants W. somnifera O. sanctum W. somnifera O. sanctum at two different sites in Karaiya Village Jaitpur Village different soil depth

9 0-10 10-20 20-30 30-40 Soil Depth (cm) 8 7 6 5 4 Soil pH 3 2 Fig. 8: Soil pH of 1 rhizosphere soil of test 0 medicinal plants at two W. somnifera O. sanctum W. somnifera O. sanctum different sites in Kariaya Village Jaitpur Village different soil depth

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DISCUSSION In the present study, the rhizosphere of two medicinal plants viz. Withania somnifera and Ocimum sanctum in different soil depth at two locations showed common as well as variant VAMF flora. Such variations in the VA mycorrhizal fungal community at different rhizosphere zone of plants have been reported earlier (Jakobsen and Nielsen, 1983; 1986; Thompson, 1991; and Oehl et al., 2005). We investigated the rhizosphere soil over a depth range from surface to 40 cm depth. As expected from 0 to 20 cm depth the rhizosphere of both the plants contained the greater VA mycorrhizal fungal spore populations. Ecological studies on the community structure of arbuscular mycorrhizal fungi are generally restricted to the main rooting zone from 10 to 25 cm soil depth (Douds et al., 1995; Guadarrama and Alvarez-Sanchez, 1999; and Bever et al., 2001). Data from both the site considered together, it was found that the fungal community composition changed with the soil depth, VA mycorrhizal fungal spore population were found decreasing with increasing soil depth. These data compliment the observations of Oehl et al. (2005) that VAM spore abundance and species richness decreased with increasing soil depth. Few studies also support, which done in the subsoil that increasing soil depth, a decrease was found in the percentage of roots colonized by AMF (Jakobsen and Nielsen, 1983; Rillig and Field, 2003), in the number of infective propagules (An et al., 1990), and in the amount of extra radical AMF hyphae (Kabir et al., 1998). In the present study maximum number of morphotypes as well as maximum percent population of spores was recorded under the genus Glomus. The genus Glomus is reported to be the dominant VAM fungi in some of the forest ecosystems (Sharma et al., 1986; Tamuli and Boruah, 2002). Vyas and Soni, 2004; Vyas et al., 2006; have reported dominance of Glomus from Sagar. Dwivedi et al., 2004, suggested physico chemical properties of soil of Sagar are responsible for the occurrence of differential VAMF. Here, many species were recorded in low numbers that too in one of the samplings only in the test sites. The rarity of some species may be an account of their narrow adaptability in contrast to Glomus species, which showed adaptability. Schenck and Kinloch (1980) attributed the abundance of Glomus species in the soils to their wide adaptability to different plants and environmental conditions. Many species of VA mycorrhizal fungi were frequently found in the Jaitpur village. Interestingly, these species does not found in the Karaiya village soil such as A. foveata, A. mellea, A. scrobiculata, E. infrequens, G. claroideum, G. etunicatum, and G. macrocarpum. However, their number decreases along with increasing soil depths. It is assumed that these VA mycorrhizal fungi, at least in central India

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 102 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 preferentially inhabit undisturbed topsoil, rich in organic matter as occurring in Jaitpur village is as a good example. Another possibility is that they might need specific plant hosts. Differences in VA mycorrhizal species in the rhizosphere region with two plants growing in two different soils may be attributed to the physico-chemical properties of both the soils. It is deduced from the results that soil of Jaitpur village is a natural soil, loamy in structure. Therefore, does not retain water, because pore size of soil particles is bigger which provide enough space for spores and mycelium to proliferate even in deeper zones. In contrast to the Karaiya village soil is a mixed soil having loam and clay 1:1 combination hence, it does not provides adequate space to VAMF spores to generate/proliferate. Since, a clay soil particle has capacity to retain water, therefore moisture content in the soil remains for larger duration, which resulted in to poor occurrence of VAMF. Wet conditions are known for their deleterious effect on VAMF population (Dubey, 2006). A. nicolsonii, Arch. Gerdemannii, G. clarum, G. fasciculatum, G. heterosporum, G. hoi, G. intraradices, and G. mosseae are frequently found in different rhizosphere zone with both the plants at both the sites. Oehl et al., (2003) called this type of VAMF species as AMF 'generalists' or even AMF 'weed' species (JPW Young Pers.com). We assume that even these AMF 'generalists' might fulfil different ecological functions. E. infreuens and S. pellucida in particular associated with O. sanctum were found to occur more abundantly with increasing soil depth. Thus at least with respect to spore formation, these species appear to be specialized for deeper layers of the soils. This observation agrees with earliest findings of Mader et al., 2002; Jansa et al., 2003; and Oehl et al., 2004. The occurrence of S. calospora and S. pellucida spore were found to be negative correlated with soil contents of available phosphorous (Oehl et al., 2004). These findings suggest these possible reasons for the stimulation of development of S. pellucida in deeper soil layers, mainly the reduced mechanical soil disturbances and this effect to decreased supply of phosphorous. In the present study there was highly negative significant correlation observed between soil parameters and fungal spore density in the samplings. The ability of the soil to support mycorrhizal population significantly decreases with increasing soil depth and is no doubt, greatly influenced by the total number of VA mycorrhizal propagules at a given depth. The average VA mycorrhizal spore population approaches zero at increased soil depths. Linear regression is a reasonably accurate statistical model for the data. However, mycorrhizae are absent at the soil surface, where there are no roots, yet linear models have a 'Y' intercept at zero depth. In reality, VA mycorrhizal spore population should be zero at the soil surface (zero depth), so

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 103 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 linear models do not account for the absence of mycorrhizae at the soil surface. The use of narrow soil profiles (1-2 cm) for estimating fungal population could be a solution for developing a biological, nonlinear model that reflects the actual ability of the soil to support mycorrhizal formation. Fibrous root systems such as those found in W. somnifera decrease with increasing soil depth. Data from cultivated soil (Sutton and Barron, 1972; Smith, 1978), from grassland soil (Sparling and Tinker, 1975), and from semi-arid soil (Schwab and Reeves, 1981) also support our results. These observations strongly support Redhead's (1977) conclusion that VAM decrease markedly below 15 cm and are consistent with similar observations of Warcup (1951) for saprobic fungi. Mycorrhiza and fungal propagules of VAMF may occur at much greater depths in soil than those depths that we examined. It was found both colonization percent and intensity decreased with increasing depth in Tall grass or True prairie species, but Glomus fasciculatum was associated with forbs roots at depths to 220 cm. These results suggest that spore viability may vary with soil moisture, and spore germination may occur at soil moisture levels that are not optimal for plant roots. Our data support previous observation of Trinick (1977) that the amount of moisture initially present in soil may affect mycorrhizal colonization of roots and thus the fungal spore density of soil. It was also observed that a significant linear relationship between moisture initially present in the soil and VA mycorrhizal spore population. Spore density of VA mycorrhizal fungi inversely propositional to moisture therefore losses the VAMF. Though relationship between soil moisture and spore population is highly significant relationship, get overriding factor is depth this can be justified simply by fewer roots, fewer mycorrhiza and fewer propagules in collected soil from lower depths. Survival of VA mycorrhizal fungi and subsequent spore germination may depend on a species' adaptation and on the influence of physical parameters of the soil such as pH (Green et al., 1976). Friese and Koske (1991) found no significant correlation between VA mycorrhizal fungal spore clumping and soil pH. Bagyaraj (1991) points out that the interpretation of a pH effect on VAM fungal spore germination is difficult because many chemical properties of soil vary with changes in pH. Soil pH over a range of 4.8-8.0 significantly influenced germination of Glomus epigaeum Daniels & Trappe spores; optimum germination occurred at pH 7 (Daniels and Trappe, 1980). The regression analysis of the VAMF spore population of the rhizosphere soil of test plants and soil pH shows a significant relationship. Spore density decrease as soil pH increases. Our results indirectly support Powell and Bagyaraj's (1984) conclusion that pH can influence spore germination in VAM fungal species, and that spore germination occurs within a range that is acceptable for plant

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 104 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 growth. In spite of the significant relationship between soil pH and fungal population, I believe, again, the overriding factor is depth. The soil pH range covers less than one order of magnitude. As depth increases, there are fewer propagules to contribute to mycorrhizal population. Direct cause and effect relationships between soil moisture or pH and mycorrhizal formation are equivocal. Peat and Fitter (1993) found no relationship between soil moisture and frequency of mycorrhizal colonization for British plants, and they reported that VAM occur at greater maximum soil pH values (ca. 6.0) than do ecto- or ericoid mycorrhizae. Soil from our study site ranged from pH 6.0 to 7.5. The occurrence of VAM at selected sites are consistent with the reports of Peat and Fitter (1993) and Read (1989). We conclude that soil pH has little direct effect on mycorrhizal population. Further Wang et al., 1993 had also reported field observations in Britain that percentage colonization and crop yield were little affected by soil pH ranging from 4.5 to 7.5. This study shows that the frequency of genera and species of VA mycorrhizal fungi isolated from both the site varied with the above ground vegetation and with changes in soil moisture and soil pH. Currently, we have limited means for accurately determining the complex of genera and species that forming symbiosis with host plants in natural soil and that are responsible for variations in fungal density obtained from soil samples. Recent advancements in characterizing mycorrhizae with molecular markers will greatly improve our understanding of the ecology of these fungi. ACKNOWLEDGEMENT Authors are thankful to Head, Department of Botany, Dr. H.S. Gour University, Sagar, MM thankfully acknowledge UGC for financially assistance. REFERENCES  Abbott, L. & A.Robson. 1991. Factors influencing the occurrence of vesicular- arbuscular mycorrhizas. Agric Ecosyst Environ 35: 121–150  An, Z.Q., J.H. Grove, J.W. Hendrix, D.E. Hershman & G.T. Henson .1990. Vertical distribution of endogonaceous mycorrhizal fungi associated with soybean, as affected by soil fumigation. Soil Biology and Biochemistry 22: 715-719.  Bagyaraj D.J. 1991. Ecology of vesicular-arbuscular mycorrhizae. In: Arora DK, Rai B, Mukerji KG, Knudsen GR. eds. Handbook of applied mycology: Soil and plants vol. I. Marcel Dekker, Inc., New York, New York. Pp. 3-34.

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 Bever, J., J.Morton, J.Antonovics & P.Schultz .1996. Host-dependent sporulation and diversity of arbuscular mycorrhizal fungi in a mown grassland. J Ecol 84:71–82  Bever, J.D., P.A. Schultz, A.Pringle & J.B. Morton. 2001. Arbuscular mycorrhizal fungi: more diverse than meets the eye, and the ecological tale of why. Bioscience 51: 923-931.  Brown, A.& C.Bledsoe. 1996. Spatial and temporal dynamics of mycorrhizas in Jaumea carnosa, a tidal saltmarsh halophyte. J Ecol 84:703–715  Caravaca, F., M.M. Alguacil, J.M. Barea & A.Roldan. 2005.Survival of inocula and native AM fungi species associated with shrubs in degraded Mediterranean ecosystem. Soil Biol. Biochem. 37:227-233  Daniels, B.A. & J.M. Trappe. 1980. Factors affecting spore germination of the vesicular-arbuscular mycorrhizal fungus, Glomus epigaeus. Mycologia 72: 457-471.  Douds, D.D., L. Galvez, R.R. Janke & P.Wagoner. 1995. Effects of tillage and farming systems upon populations and distribution of vesicular arbuscular mycorrhizal fungi. Agriculture, Ecosystems and Environment 52: 111-118.  Dubey, A. 2006. Studies on diversity of AM fungi with special reference to rice crop. Ph.D. Thesis, Dr. H. S. Gour University, Sagar (M.P.) India.  Dwivedi, O.P.,, R.K. Yadav, D. Vyas & K.M. Vyas. 2004. Role of potassium on the occurrence of vesicular arbuscular mycorrhizal spores in the rhizosphere of Lantana sp. In; Microbiology and Biotechnology for sustainable developments (Ed.) P.C. Jain, CBS Publishers and distributors, New Delhi 248-253.

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 Govindarajulu, M., P.Pfeffer, H.R. Jin, J. Abubaker, D.D. Douds, J.W. Allen, H. Bucking, P.J. Lammers& Y.Shachar-Hill .(2005). Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature 435: 819-823.  Green, N.E., S.O. Graham & N.C.Schenck. 1976.The influence of pH on the germination of vesicular-arbuscular mycorrhizal spores.Mycologia 68: 929- 934  Grime, J.P., J.M. Mackey, S.M. Hillier & D.J. Read. 1987. Floristic diversity in a model system using experimental microcosms.Nature 328:420–422  Guadarrama, P. & F. Alvarez-Sanchez .1999. Abundance of arbuscular mycorrhizal fungi spores in different environments in a tropical rain forest, Veracruz, Mexico. Mycorrhiza 8: 267-270.  Gupta S.C.& V.K. Kapoor. 1997. Fundamentals of applied statistics (III ed) S Chand and Sons. New Delhi.  Hartnett, D.& G. Wilson. 1999. Mycorrhizae influence plant community structure and diversity in tallgrass prairie. Ecology 80:1187–1195  Hodge, A., C.D. Campbell, & A.H. Fitter. (2001). An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material. Nature 413: 297-299  Ingham, E. & M. Wilson .1999. The mycorrhizal colonization of six wetland species at sites differing in land use history. Mycorrhiza 9:233–235  Jackson M.L. 1967. Soil Chemical Analysis. Prentice Hall of India (Ltd.) New Delhi, pp. 428.  Jakobsen, I. & N.E. Nielsen. 1983. Vesicular arbuscular mycorrhiza in field- grown crops I. Mycorrhizal infection in cereals and peas at various times and soil depths. New Phytologist 93: 401-413.  Jansa, J.A., A. Mozafar, T. Anken, R. Ruh, I.R. Sanders & E. Frossard .2003. Soil tillage affects the community structures of mycorrhizal fungi in maize roots. Ecological Applications 13: 1164-1176.  Johnson, N.C., D.Tilman& D. Wedin .(1992). Plant and soil controls on mycorrhizal fungal communities. Ecology 73:2034–2042  Kabir, Z., I.P. O'Halloran, P. Widden & C.Hamel .1998. Vertical distribution of arbuscular mycorrhizal fungi under corn (Zea mays L.) in no-till and conventional tillage systems. Mycorrhiza 8: 53-55.

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 Lugo M.A.& M.N. Cabello. 2002. Native arbuscular mycorrhizal (AMF) from mountain grassland (Córdoba, Argentina) I. Seasonal variation of fungal spore diversity. Mycologia 94:579–586  Mader, P., A. Fliessabach, D. Dubois, L. Gunst, P. Fried & U. Niggli. 2002. Soil fertility and biodiversity in organic farming. Science 296: 1694-1697.  Miller, S.& J. Bever .1999. Distribution of arbuscular mycorrhizal fungi in stands of the wetland grass Panicum hemitomon along a wide hydrologic gradient. Oecologia 119:586–592  Mosse, B., D. Stribley & F. Le Tacon. 1981. Ecology of mycorrhizas and mycorrhizal fungi. In: Alexander M (ed) Advances in microbial ecology. Plenun Press, New York, pp 137–210  Mosse, B., D.P. Stribley & F. Tacon . (1981).Ecology of mycorrhizae and mycorrhizal fungi  Oehl, F., E. Sieverding, K. Ineichen, E.A. Ris, T. Boller & A. Wiemken. 2005. Community structure of arbuscular mycorrhizal fungi at different soil depths in extensively managed agroecosystems.NewPhytologist 165: 273-283.  Oehl, F., E. Sieverding, K. Ineichen, P. Mader, D Dubos, T Boller & A. Wiemken .2004. Impact of long-term conventional and organic farming on the diversity of arbuscular mycorrhizal fungi. Oecologia 138: 574-583  Oehl, F., E. Sieverding, K. Ineichen, P. Mader, T. Boller & A. Wiemken. 2003. Impact of land use intensity on the species diversity of arbuscular mycorrhizal fungi in agroecosystems of central Europe. Applied and Environmental Microbiology 69: 2816-2824.  Peat, H.J.& A.H. Fitter.1993. The distribution of arbuscular mycorrhizas in the British flora. New Phytologist 125: 845- 854.  Powell, C.L. & D.J. Bagyaraj 1984. VA-Mycorrhiza. CRC Press, Boca Raton, Florida. pp. 234.  Quilambo, O.A. (2003). The vesicular- arbuscular mycorrhizal symbiosis. African J.Biotechnol. 2:539-546  Read D.J. 1989. Mycorrhizas and nutrient cycling in sand dune ecosystems. Proceedings of the Royal Society of Edinburgh, 96: 89-110.  Redhead J.F. 1977. Endotrophic mycorrhizas in Nigeria: Species of the Endogonaceae and their distribution. Transactions of the British Mycological Society 69: 275-280.

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exposed to elevated atmospheric CO2 as a function of soil depths. Plant and soil 254: 383-391.  Schenck N.C.& Y. Perez. 1987. Manual for identification of VAM fungi. Synergistic Pub. Gainesville. Fl., U.S.A.  Schenck, N.C., & R.A. Kinloch 1980. Incidence of mycorrhizal fungi on six field crops in monoculture on a newly cleared woodland site. Mycologia 72: 445-455.  Schwab, S. & F.B. Reeves .1981. The role of endomycorrhizae in revegetation practices in the semi-arid West. III. Vertical distribution of vesicular- arbuscular (VA) mycorrhiza inoculum potential. American Journal of Botany 68: 1293-1297.  Sharma, S.K., G.D. Sharma & R.R.Mishra. 1986. Status of mycorrhizae in sub-tropical forest ecosystem of Meghalaya. Acta Botanica Indica 14: 87-92.  Smith T.F. 1978. A note on the effect of soil tillage on the frequency and vertical distribution of spores of vesicular-arbuscular endophytes. Australian Journal of Soil Research 16: 359- 361.  Smith, M., D.Hartnett & G.Wilson. 1999. Interacting influence of mycorrhizal symbiosis and competition on plant diversity in tallgrass prairie. Oecologia 121:574–582  Sparling, G.P. & P.B.Tinker 1975. Mycorrhizae in Pennine grassland. In: Sanders FE, Mosse B, Tinker PB. eds. Endomycorrhizae., Proceedings of a Symposium held at the University of Leeds, 22–25 July 1974, Academic Press, New York, New York, Pp. 545-560.  Sutton, J.C. & G.L. Barron .1972. Population dynamics of Endogone spores in soil. Canadian Journal of Botany 50: 1909-1914.  Tamuli, P. & P.Boruah. 2002. VAM association of agar wood tree in Jorhat district of the Brahmaputra valley. Indian Forester 128 (9): 991-994.  Thompson J.P. 1991. Improving the mycorrhizal infection of the soil through cultural practices and effects on growth and phosphorous uptake by plants. In: Johansen C, Lee KK, Sahrawat KL, eds. Phosphrous nutrition of grain legumes in the semi-arid tropics. Pantancheru, India: International Crops Research Institutes for the Semi-arid Tropics, 117-138.  Trinick M.J. 1977. Vesicular arbuscular infection and soil phosphorus utilization in Lupinus spp. New Phytologist 78: 297-304.

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 Van der Heijden, M.G.A., J.N. Kilronomos, M.Ursic, P.Moutoglis, R.Streitwolf-Engel, T.Boller, A.Wiemken& I.R. Sanders. 1998.Mycorrhizal fungal diversity determines plant biodiversity,  Vyas, D., & A.Soni 2004. Diversity and distribution of VAMF in the seminatural grassland. Indian Journal of Ecology 31: 170-171  Vyas, D., A. Dubey, A. Soni, M.Mishra & P. Singh. (2007). Arbuscular mycorrhizal fungi in early land plants. Mycorrhiza News.19(2)  Vyas, D., P. Singh, M. Mishra, & A. Dubey. (2008). VA Mycorrhizal Association in Weeds of Seminatural Grassland of Sagar. Indian J. Agroforestry 10(2): 91-97  Vyas. D., A. Dubey, P.K.Singh, M.K. Mishra, A. Soni & P.Soni .2006a. VA mycorrhizal fungi in tropical monsoonic grassland. Journal of Basic and Applied Mycology 5 (I&II): 78-81.  Wang, G., D.P. Stribley, P.B. Tinker & C.Walker. 1993. Effects of pH on arbuscular mycorrhiza I. Field observations on the long-term liming experiments at Rothamstead and Woburn. New Phytologist 124: 465-472.  Warcup J.H. 1951. Soil-streaming: a selective method for the isolation of fungi of Ascomycetes from soil. Transactions of the British Mycological Society 34: 515-518.

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Gymnema sylvestre R. Br- A Medicinal herb Laxmi Ahirwal, Siddhartha Singh and Archana Mehta Lab of Plant Biotechnology, Department of Botany, School of Biological and Chemical Sciences, Dr. H.S. Gour University, Sagar (M.P), India- 470 003 E-mail: [email protected] Abstract Gymnema sylvestre R.Br. commonly known as gudmar belongs to the family Asclepiadaceae. It is a potent antidiabetic plant used in folk, ayurvedic and homeopathic systems of medicine. The fresh leaves when chewed have the remarkable property of paralysing the sense of taste for sweet and bitter substance for some time because of this property it is commonly known as ‘gudmar’. Its root and Leaves have medicinal properties. This review will try to put forth an overall idea about the plant as well as present the pharmacological activities of the plant. Keywords: Gymnema sylvestre, Asclepiadaceae, medicinal importance, pharmacological activities. Classification: Kingdom : Plantae Division : Angiospermae Class : Dicotyledoneae Order : Contortae Family : Asclepiadaceae Genus : Gymnema Species : sylvestre Fig.1. Gymnema sylvestre: woody climber. Gymnema sylvestre R. Br (Asclepiadaceae) is a well-known antidiabetic plant it is also widely used to reduce obesity. It is commonly known as Meshashringi, Madhunashini, Gurmar (Bone, 2002), Merasing, Kavali, Kalikardori, Vakundi and Wood of periploca. The plant is native to central and western India, tropical Africa and Australia and distributed in Asia, Tropical Africa, Malaysia and Srilanka (Gurav et al., 2007). It used as a stomachic, diuretic and anti-diabetic remedy. The active compound of the plant is a group of acids termed as gymnemic acids. It has been observed that there could be a possible link between obesity, Gymnemic acids and diabetes (Kanetkar et al., 2007). The total saponin fraction of the leaves, commonly known as gymnemic acid, has an anti-sweetening effect (Wen-cai et al., 2000).

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Botanical Description It is perennial large climber, rooting at nodes. Leaves are elliptic, acuminate, base acute to acuminate, glabrous above sparsely or densely tomentose beneath. Flowers are small, in axillary and lateral umbel like cymes, pedicels long; Calyx-lobes long, ovate, obtuse, and pubescent; Corolla pale yellow campanulate, valvate, corona single, with 5 fleshy scales. Scales are adnate to throat of corolla tube between lobes; Anther connective produced into a membranous tip, pollinia 2, erect, carpels 2, unilocular; many ovules present in locule; Follicle is long and fusiform (Keshavamurthy and Yoganarasimhan, 1990). Phytochemistry Plant constituents include two resins (one soluble in alcohol), six percent Gymnemic acids, saponins, stigmasterol, quercitol, and the amino acid derivatives betaine, choline, and trimethylamine (Kapoor, 1990). Leaves contain lupeol, β- amyrin, stigmasterol, pentriacontane, hentricontane, α and β chlorophyll, resin, tartaric acid, gymnemic acid (anti sweet compounds) the mixture of triterpene saponins, anthraquinone derivatives, alkaloids, betain, choline and trimethylamine (Kokate, 2006). According to recent reports gymnemic acid formulations have also been found useful against obesity (Yoshikawa et al., 1997). The triterpenoid saponin contain several acylated (tigloyl, methylbutyroyl etc.) derivatives of deacylgymnemic acid which is 3-O-b-glucuronide of gymnemagenin (3b, 16b, 21b, 22a, 23, 28-hexahydroxy-olean-12-ene). The individual gymnemic acids (saponins) include gymnemic acids I-VII, gymnemosides A-F, gymnemasaponins etc. (Gurav et al., 2007). Plant part used: Leaves and roots Medicinal Properties The plant is reported to be useful in inflammations, hepatosplenomegaly, dyspepsia, constipation, haemorrhoids, helminthiasis, cough, asthma, bronchitis, cardiopathy, jaundice, intermittent fever, piles (Sharma, 1983), amenorrhea, conjunctivitis, leucoderma, and urinary disorders (Nadkarni, 1993). Its root is used to cure snakebite. The fresh leaves when chewed have the remarkable property of paralysing the sense of taste for sweet and bitter substance for some time (Warrier et al., 1995). Leaves (triturated and mixed with castor oil) are applied to swollen glands and enlargement of internal viscera as the liver and spleen (Nadkarni, 1954). According to Ayurvedic literature leaves were used as a cardiotonic, diuretic (Reddy et al., 2004), laxative, stimulant, stomachic and uterine tonic and these are also used as antiviral, diuretic, antiallergic, hypoglycemic, hypolipidemic, for the treatment of obesity and dental caries (The Ayurvedic Pharmacopoeia of India, 2006). The fruits

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 112 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 are bitter and carminative and have been used to treat leprosy, diabetes, bronchitis, worms, ulcers and poisoning. The whole plant is taken orally in dysentery. The plant is used as a constituent of many traditional Ayurvedic preparations including ayaskrti, varunadi kasayam and varunadi ghrtam. Leaves are commonly used in diabetes (Sivaprakasam et al., 1984). Pharmacological activities Gymnemic acid formulations have also been found useful against obesity, according to recent reports (Yoshikawa et al., 1993). The aqueous extract of G. sylvestre leaves (GSE) tested on various inflammatory models and showed anti- inflammatory activity by significantly inhibiting carrageenan-induced rat paw oedema and peritoneal ascites in mice (Diwan et al., 1995). Investigation of the hypoglycemic activity of saponin constituents from gymnemic acid, a crude saponin fraction of G. sylvestre, identified not only two new saponins, gymnemosides a and b, but also gymnemic acid V as the active principle (Murakami et al., 1996). Recently, effects of the water soluble fraction of an alcoholic extract of G. sylvestre leaves on glycogen content of isolated rat hemidiaphragm was studied in normal and glucose fed hyperglycaemic rats. In glucose fed rats, the leaf extract lowered the glycogen content of the tissue and this effect was amplified by insulin (Chattopadhyay, 1998). The aqueous extract of G. sylvestre leaves tested on various inflammatory models showed anti-inflammatory activity by significantly inhibiting carrageenan-induced rat paw oedema and peritoneal ascites in mice (Diwan et al., 1995; Malik et al., 2008). The anticancer-cytotoxic activities of isolated saponins, gymnemagenol, from G. sylvestre leaves, were tested under in vitro conditions in HeLa cells. The gymnemagenol showed a good anticancer-cytotoxic activity on HeLa cells under in vitro conditions (Khanna and Kannabiran, 2009). The Gymnema sylvestre alcoholic leaf extract showed antioxidant activity by inhibiting DPPH, scavenging superoxide and hydrogen peroxide. It also showed reducing power ability in ferric reducing model (Rachh et al., 2009). It was investigated that treatment with hydroalcoholic extract of Gymnema sylvestre R. Br. leaves significantly decreased total ser un cholesterol, triglycerides, low density lipoproteins, very low density lipoprotein and increased the high density lipoproteins in hyperlipidemic rats (Rachh et al., 2010). The anthelmintic potential of various extract of Gymnema sylvestre was evaluated taking Indian adult earthworms as models (Pheretima posthuma). The methanolic extract of G. sylvestre was found to be more potent (paralysis 2.66 ± 0.30min, death 5.83 ± 0.30min) at a concentration of 40mg/ml (Ahirwal et al., 2010). Similarly, the antimicrobial activity of the methanol and aqueous extracts of leaves of Gymnema sylvestre was evaluated using agar diffusion method (well method). It was found that aqueous extract revealed significant activity against

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 113 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Cladosporium oxysporum (6mm at 800 µg/ml) while methanol extract showed significant activity against E.coli (6mm at 800 µg/ml). Both the extracts showed concentration dependent activity (Ahirwal et al., 2012). Ahirwal et al., (2013) investigated the in vitro antioxidant potential of methanolic extract of Gymnema sylvestre leaves by using DPPH, hydroxyl radical, nitric oxide radical scavenging as well as ferric reducing power assays. The extract showed significant activities in a concentration dependent manner when compared with standard drug Butylated hydroxy anisole. Results revealed that the methanolic extract of G. sylvestre showed significant antioxidant activity. Conclusion All the above pharmacological investigation reports and medicinal properties evidence that a Gymnema sylvestre is a potent medicinal herb and has promising future prospective in the scientific field of health and medical research. References 1. Ahirwal L, Mehta A, Mehta P, John J, Singh S (2010). Anthelmintic Potential of Gymnema sylvestre and Swertia chirata. Inventi Rapid: Ethanopharmacol, 1(2): 9-10. 2. Ahirwal L, Singh S, Mehta A, Rajoria A (2012). Evaluation of Antimicrobial Potential of Gymnema sylvestre Leaves Extracts. Int J Pharm Sci Rev Res, 16(2): 43-46.

3. Ahirwal L, Singh S, Hajra S, Mehta A (2013). Screening of Methanolic Extract of Gymnema Sylvestre R. Br. leaves for Antioxidant Potential. International Journal of Pharmaceutical Sciences Review and Research. Int J Pharm Sci Rev Res, 19(1): 87-91. 4. Bone K (2002). Official home page of Gymnema: A key herb in the management of diabetes. Retrieved March 18,2007, from http://www.townsendletter.com. 5. Chattopadhyay RR (1998). Possible mechanism of antihyperglycaemic effect of Gymnema sylvestre leaf extract, part I. Gen Pharmacol, 31: 495-496. 6. Diwan PV, Margaret I, Ramakrishna S (1995). Influence of Gymnema sylvestre on inflammation. Inflammopharmacology, 3(3): 271-277. 7. Diwan PV, Margaret I, Ramakrishna S (1995). Influence of Gymnema sylvestre on inflammation. Inflammopharmacology, 3(3): 271-277.

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8. Gurav S, Gulkari V, Duragkar N, Patil A (2007). Systemic review Pharmacognosy, phytochemistry, pharmacology and clinical applications of Gymnema sylvestre R. Br. Pharmacog Rev, 1: 338-343. 9. Joshi SG (2000). (ed.), Medicinal Plants, Oxford and IBH, New Delhi: 216.

10. Kanetkar P, Singhal R, Kamat M (2007). Gymnema sylvestre: A Memoir. J Clin Biochem Nutr, 41(2): 77–81. 11. Kapoor LD (1990). Handbook of Ayurvedic Medicinal Plants. Boca Raton, FL: CRC Press, Inc; 200-201. 12. Keshavamurthy KR, Yoganarasimhan SN (1990). "Flora of Coorg - Karnataka"; Vimsat Publishers, Bangalore; 282. 13. Khanna VG and Kannabiran K (2009). Anticancer-cytotoxic activity of saponins isolated from the leaves of Gymnema sylvestre and Eclipta prostrata on HeLa cells. International J Green Pharmacy, 3(3): 227-229. 14. Kokate CK, Purohit AP, Gokhale SB (2006). Pharmacognosy, Nirali Prakashan, Pune, 36th Edition: 252. 15. Malik JK, Manvi FV, Alagawadi KR, Noolvi M (2008). Evaluation of anti- inflammatory activity of Gymnema sylvestre leaves extract in rats. International J Green Pharmacy, 2(2): 114-115. 16. Murakami C, Murakami T, Kadoya M (1996). New hypoglycaemic constituents in gymnemic acid from Gymnema sylvestre. Chem Pharm Bull, 44: 469-471. 17. Nadkarni AK (1954). Indian Materia Medica, Popular Prakashan, Bombay. 18. Nadkarni KM (1993). Indian Materia Medica , Popular Prakashan, Bombay; 1: 596-599. 19. Rachh PR, Patel SR, Hirpara HV, Rupareliya MT, Rachh MR, Bhargava AS,

Patel NM, Modi DC (2009). In vitro evaluation of antioxidant activity of Gymnema sylvestre R. Br. leaf extract. Rom J Biol - Plant Biol, 54 (2): 141– 148. 20. Rachh PR, Rachh MR, Ghadiya NR, Modi DC, Modi KP, Patel NM, Rupareliya MT (2010). Antihyperlipidemic Activity of Gymenma sylvestre R. Br. Leaf Extract on Rats fed with high cholesterol diet. International Journal of Pharmacology, 6 (2): 138-141. 21. Reddy S, Gopal G, Sit G (2004). In vitro multiplication of Gymnema sylvestre R Br: An important medicinal plant. Curr Sci, 10: 1-4.

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22. Sharma, PV (1983). Dravyaguna Vijnana, Varanasi (in Hindi). 23. Sivaprakasam K, Rao KK, Yashodha R, Velichamy G (1984). Remedy for Diabetes Mellitus. J Res Ayur & Siddha, 5(1-4): 25-32. 24. The Ayurvedic Pharmacopoeia of India (2006). Government of India, Ministry of Health and Family Welfare Department of Indian System of Medicine and Homoeopathy, New Delhi, Part-I, 1st Edition; 5: 110-114, 123-124. 25. Wan-cai Y, Zhang Q, Liu X, Che C, Zhao S (2000). Oleanane saponins from Gymnema sylvestre. Phytochemistry, 53: 893-899. 26. Warrier, PK, Nambiar, VPK, Ramankutty C (1995). Indian Medicinal Plants. Orient Longman Ltd., Madras; l: 1-5. 27. Yoshikawa K, Kondo Y, Arihara S, Matsuura K (1993). Antisweet natural products IX structures of gymnemic acids XV-XVIII from Gymnema sylvestre R. Br. Chem Pharm Bull, 41: 1730–1732. 28. Yoshikawa M, Murakami T, Kadoya M (1997). Medicinal food stuffs. IX. The inhibitors of glucose absorption from the leaves of Gymnema sylvestre R. Br. (Asclepiadaceae): Structures of gymnemosides A and B. Chem Pharm Bull, 45: 1671-1676.

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An updated commentary on the role of RNA interference in the plant disease management

Deepak Singh Bagri and Chandrama Prakash Upadhyaya* School of Biological Sciences, Department of Biotechnology, Dr. H. S. Gour Central University, Sagar-470003, M.P., INDIA *Corresponding author: Email: [email protected], Ph: +91-7587194330 Abstract A vast number of plant pathogens from viroids of a few hundred nucleotides to higher plants cause diseases in our crops. Their effects range from mild symptoms to catastrophes in which large areas planted to food crops are destroyed globally. Plant pathogens are difficult to control because their populations are variable in time, space, and genotype. Though plant breeding has been the classical means of manipulating the plant genome to develop resistant cultivar for controlling plants diseases, however, the advances in genetic engineering provides a novel approach being pursued to render plants resistant to plant pathogen ranging from fungi, bacteria, viruses and nematodes. RNA interference (RNAi) which is a homology- dependent gene silencing technology that is initiated by double stranded RNA (dsRNA) has emerged as a genetic tool for engineering plants resistance against various plant pathogens. Silencing specific genes by RNAi has been proved to be a powerful natural tool in order to overcome this problem, as the disease resistant transgenic plants can be produced within a regulatory framework. Recent studies have been successful in developing potent silencing effects by using target double-stranded RNAs through an efficient vector system. Transgenic plants expressing RNAi vectors as well as dsRNA sprays on the crop plants have been successful for efficient control of plant pathogens. Present review discusses the strategies and applications of this novel technology in plant disease management for sustainable crop productivity. Key words: RNA interference RNAi, crop resistance, biotechnology, nematode, insect, parasitic weed, fungus. Introduction To offset the crop losses from pathogens, various attempts have been made in the field of disease management since inception of green revolution. Chemicals have been traditionally used on crop plants to prevent crop damages. Once chemicals were discovered to pollute the environment and be harmful to human health, agriculture research began to focus on alternative safer means. During last two and half decades much attention has been paid for integrated disease management practices which make it inexpensive and safe method for disease control (Mandal et al. 2012). Though, plant breeding has been the classical means of manipulating the plant

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 117 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 genome to develop resistant cultivar for controlling plants diseases. Further, study of genetic host resistance fulfils this requirement but is a continuous endeavor as the boom and bust cycle goes on in the process of co-evolution, though therapeutic tools based on current molecular biology hold the key after the exploitation of traditional breeding and biotechnological methods like use of molecular marker for identification, mapping, cloning of pest and disease resistant genes and their utilization by introgression, pyramiding and development of transgenics (Mann et al. 2008, Wani et al. 2010, Sanghera et al. 2011). The inherent risks associated with traditional transgenics can be mitigated by new and innovative strategies and transgenic plants can be produced within a regulatory framework (Sanghera et al. 2010). RNA interference is a natural process which silences specific genes before being translated. RNAi inducers, in the form of transgenic plants or a crop spray, have the potential to effectively silence specific genes (Baum et al. 2007, Mao et al. 2007). Both techniques have been successful in silencing genes. During the last decade our knowledge repertoire of RNA-mediated functions has hugely increased with the discovery of small non-coding RNAs which play a central part in a process called RNA silencing. Ironically the very important phenomenon of co-suppression has recently been recognized as a manifestation of RNA interference (RNAi) an endogenous pathway for negative post-transcriptional regulation. RNAi has revolutionized the possibilities for creating custom “knock-downs” of gene activity. RNAi operates in both plants and animals and uses double stranded (dsRNA) as a trigger that targets homologous mRNAs for degradation or inhibiting its transcription or translation (DeBakker et al. 2002; Almeida and Allshire 2005; Sanghera et al. 2010) whereby susceptible genes can be silenced. This RNA-mediated gene control technology has provided new platforms for developing eco-friendly molecular tools for crop improvement by suppressing the genes responsible for various stresses and improving novel traits in plants including disease resistance and will be a promising future therapeutic agent to combat plant invaders. It has emerged as a method of choice for gene targeting in fungi (Nakayashiki 2005) viruses (Baulcombe 2004) bacteria (Escobar et al. 2001) and plants (Brodersen and Voinnet 2006) as it allows the study of the function of hundreds of thousands of genes to be tested (Godge et al. 2008). It can silence a gene throughout an organism or in specific tissues offer the versatility to partially silence or completely turn off genes. Transgenic plants would be cost-effective by producing RNAi inducers throughout a plant’s life constantly silencing different pathogen genes. Therefore, the applications of RNAi technology in the improvement of plants with special reference to disease management are specifically focused below in different sections of this chapter. It is important to point out that although the cosuppression phenomenon was originally observed in plants, it is not restricted to plants and has also been

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 118 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 demonstrated in metazoans and mammals (Hannon, G. J. 2002). Later on, it became clear that the expression of the transgene led to the formation of dsRNA, which, in turn, initiated PTGS. For example, in the case of co suppressed petunia plants, chsA mRNA formed a partial duplex, since there are regions of self complementarity located between chsA 3_ coding region and its 3_ untranslated region (Metzlaff, M., M. ODell, P. D. Cluster, and R. B. Flavell. 1997). This was revealed by DNA sequence analysis and experimental detection of in vitro-transcribed, RNase-resistant duplex chsA RNA. In another study, a p35S-ACC (1-aminocyclopropane-1- carboxylate [ACC] oxidase) sense transgene carrying a small inverted repeat in the 5_ untranslated region was introduced into tomato to test the role of dsRNA structure as an inducer of PTGS. Cosuppression of the endogenous acc gene occurred at a higher frequency in these plants than in those harbouring only the p35S-ACC sense transgene without the inverted repeat (Hamilton, A. J., S. Brown, H. Yuanhai, M. Ishizuka, and A. Lowe. 1998). Recent researches from several laboratories have established that the loss in steady-state accumulation of the target mRNA is almost total if the designed transgene construct of the transgenic plant produces the nuclear transcript in the duplex conformation. Very recently it was reported that the expression of self-cRNA of plum pox virus under the control of rolC promoter caused degradation of transgenic viral RNA and as a result, the systemic disease resistance to challenge inoculums of plum pox virus occurred with a high frequency in transgenic Nicotiana benthamiana (Pandolfini, T., B. Molesini, L. Avesani, A. Spena, and A. Polverari. 2003). This evidence points out that the production of dsRNA is required to initiate PTGS in plants. Based on this, plants carrying strongly transcribing transgenes in both the sense and antisense orientations are currently being produced that show strong PTGS features. These transgenic plants can silence endogene, invading viral RNA, or unwanted foreign genes in a sequence-specific and heritable manner. Generally, the sense and antisense components of the above-mentioned transgenes are separated only by an intron to increase the efficacy of PTGS (Smith, N. A., S. P. Singh, M. B. Wang, P. A. Stoutjesdijk, A. G. Green, and P. M. Waterhouse. 2000). For example, Arabidopsis thaliana and Lycopersicon esculentum (tomato) plants were transformed with a transgene construct designed to generate self-complementary iaaM and ipt transcripts. iaaM and ipt are oncogenes of agrobacteria that are responsible for crown gall formation in infected plants. The transgenic lines retained susceptibility to Agrobacterium transformation but were highly refractory to tumorigenesis, providing functional resistance to crown gall disease by posttranscriptional degradation of the iaaM and ipt transcripts (Escobar, M. A., E. L. Civerolo, K. R. Summerfelt, and A. M. Dandekar. 2001).

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 119 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

RNAi and Quelling PTGS is a breakthrough in gene silencing, but homology dependent gene silencing phenomena called quelling were also observed independently in fungal systems. Quelling came to light during attempts to boost the production of an orange pigment made by the gene al1 of the fungus Neurospora crassa (Cogoni, C., J. T. Irelan, M. Schumacher, T. J. Schmidhauser, E. U. Selker, and G. Macino. 1996). A N. crassa strain containing a wild-type al1_ gene (orange phenotype) was transformed with a plasmid containing a 1,500-bp fragment of the coding sequence of the al1 gene. A few transformants were stably quelled and showed albino phenotypes. In the al1-quelled strain, the level of unspliced al1 mRNA was similar to that of the wild- type strain, whereas the native al1 mRNA was highly reduced, indicating that quelling and not the rate of transcription affected the level of mature mRNA in a homology- dependent manner. The phenomenon of RNAi first came into the limelight following the discovery by Fire et al. (Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello. 1998), who unequivocally demonstrated the biochemical nature of inducers in gene silencing by introducing purified dsRNA directly into the body of Caenorhabditis elegans. The investigators injected dsRNA corresponding to a 742-nucleotide segment of unc22 into either the gonad or body cavity region of an adult nematode. unc22 encodes an abundant but nonessential myofilament protein, and the decrease in unc22 activity is supposed to produce an increasingly severe twitching phenotype. The injected animal showed weak twitching, whereas the progeny individuals were strong twitchers. The investigators showed that similar loss- of-function individuals could also be generated with dsRNAs corresponding to four other nematode genes. The phenotypes produced by interference by various dsRNAs were extremely specific. This experiment paved the way for easy production of null mutants, and the process of silencing a functional gene by exogenous application of dsRNA was termed RNA interference (RNAi). RNAi in C. elegans was also initiated simply by soaking the worms in a solution containing dsRNAs or by feeding the worms Escherichia coli organisms that expressed the dsRNAs (Timmons, L., and A. Fire. 1998). This is a very potent method, requiring only catalytic amounts of dsRNA per cell to silence gene expression. The silencing spread not only from the gut of the worm to the remainder of the body, but also through the germ line to several generations. These phenomena of RNAi have also been demonstrated to occur in Drosophila melanogaster and many other invertebrates and vertebrates. Gene Silencing Complex Components For the identification of various components of gene silencing complex both genetic and biochemical approaches have been undertaken. Genetic screens were carried out in the fungus Neurospora crassa, the alga Chlamydomonas reinhardtii, the

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 120 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 nematode Caenorhabditis elegans, and the plant A. thaliana to search for mutants defective in RNA interference, quelling, or PTGS. Analyses of these mutants led to the identification of host-encoded proteins involved in gene silencing and also revealed that a number of essential enzymes or factors are common to these processes. Some of the important components are as follows. Dicer These are RNase III family members among the few nucleases that show specificity for dsRNAs (Nicholson, A. W. 1999) and cleave them with 3_ overhangs of 2 to 3 nucleotides and 5_-phosphate and 3_-hydroxyl termini (Elbashir, S. M., W. Lendeckel, and T. Tuschl. 2001). Bernstein et al. (Bernstein, E., A. A. Caudy, S. M. Hammond, and G. J. Hannon. 2001) identified an RNase III-like enzyme in Drosophila extract which was shown to have the ability to produce fragments of 22 nucleotides, similar to the size produced during RNAi. These authors showed that this enzyme is involved in the initiation of RNAi. For its ability to digest dsRNA into uniformly sized small RNAs (siRNA), this enzyme was named Dicer (DCR) sometime called slicing enzyme. These nucleases are evolutionarily conserved in worms, flies, fungi, plants, and mammals. Dicer has four distinct domains: an dual RNase III motifs, aminoterminal helicase domain, a dsRNA binding domain, and a PAZ domain (a 110-amino-acid domain present in proteins like Piwi, Argo, and Zwille/Pinhead), which it shares with the RDE1/QDE2/Argonaute family of proteins that has been genetically linked to RNAi by independent studies (Tabara, H., M. Sarkissian, W. G. Kelly, J. Fleenor, A. Grishok, L. Timmons, A. Fire, and C. C. Mello. 1999). Cleavage by Dicer is catalyzed by its tandem RNase III domains. Some DCR proteins, including the one from D. melanogaster, contain an ATP-binding motif along with the DEAD box RNA helicase domain. The predicted C. elegans Dicer homologue, K12H4.8, was referred as DCR1 because it was demonstrated to be the functional ortholog of the Drosophila Dicer protein (Peele, C., C. V. Jorden, N. Muangsan, M. Turnage, E. Egelkrout, P. Eagle, L. Hanely-Bowdoin, and D. Robertson. 2001). The 8,165-bp DCR1 protein has a domain structure similar to that of the Drosophila Dicer protein. dcr1 mutants of C. elegans showed defects in RNAi of germ line-expressed genes but no effect on the RNAi response of somatic genes. These mutants were found to be sterile, suggesting the important role of this gene in germ line development apart from RNAi (Knight, S. W., and B. L. Bass. 2001). CAF1 has been identified as a Dicer homologue in A. thaliana, but it is not involved in PTGS activity. The structure of CAF1 shows the presence of the four distinct domains that were identified in the Drosophila Dicer protein (Jacobson, S. E., M. P. Running, and E. M. Meyerowitz. 1999). Dicer homologues from many different sources have been identified; recombinant Dicers have also been examined in vitro, and phylogenetic analysis of the known Dicer-like proteins indicates a common ancestry

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 121 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 of these proteins. By the complete digestion by RNase III enzyme results in dsRNA fragments of 12 to 15 bp, half the size of siRNAs (Yang, D., F. Buchholz, Z. Huang, A. Goga, C.-Y. Chen, F. M. Brodsky, and J. M. Bishop. 2002). The RNase III enzyme acts as a dimer and thus digests dsRNA with the help of two compound catalytic centers, whereas each monomer of the Dicer enzyme possesses two catalytic domains. The crystal structure of the RNase III catalytic domain was solved, and this led to the model for generation of 23- to 28-mer diced siRNA products (Blaszczyk, J., J. E. Trppea, M. Bubunenko, K. M. Routzahn, D. S. Waugh, D. L. Court, and X. Ji. 2001). In this model, the dimeric Dicer folds on the dsRNA substrate to produce four compound catalytic sites so that the two internal sites bearing partial homology lose functional significance, while the two terminal sites having the maximum homology with the consensus RNase III catalytic sequence remain active other. thus, the diced products appear as the limit digests of the RNase III enzymes and are double the size of the normal 12- to 15-mer fragments. Such a model also predicts that certain changes in Dicer structure might modify the spacing between the two active terminal sites and thus generate siRNAs of variable sizes bearing species-specific imprints. Guide Induced Silencing Complex and RISC Complex The endogenous genes of Drosophila S2 cells could be targeted in a sequence- specific manner by transfection with dsRNA, and loss-of-function phenotypes were created in cultured Drosophila cells (Hammond, S. M., E. Berstein, D. Beach, and G. J. Hannon. 2000). The inability of cellular extracts treated with a Ca2 dependent nuclease (micrococcal nuclease, which can degrade both DNA and RNA) to degrade the cognate mRNAs and the absence of this effect with DNase I treatment showed that RNA was an essential component of the nuclease activity. The sequence-specific nuclease activity observed in the cellular extracts responsible for ablating target mRNAs was termed the RNA-induced silencing complex (RISC). After p artial purification of crude extracts through differential centrifugation and anion exchange chromatography, the nuclease cofractionated with a discrete 25-nucleotide RNA species. These results suggested that small RNAs were associated with sequence- specific nuclease and served as guides to target specific messages based upon sequence recognition. In another report, the multi component RNAi nuclease was purified to homogeneity as a ribonucleic protein complex of 500 kDa (Hammond, S. M., S. Boettcher, A. A. Caudy, R. Kobayashi, and G. J. Hannon. 2001). One of the protein components of this complex was identified as a member of the Argonaute family of proteins and was termed Argonaute2 (AGO2). AGO2 is homologous to RDE1, a protein required for dsRNA-mediated gene silencing in C. elegans. AGO2 is a 130-kDa protein containing polyglutamine residues, PAZ, and PIWI domains characteristic of members of the Argonaute gene family. The Argonaute family

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 122 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 members have been linked both to the gene-silencing phenomenon and to the control of development in diverse species. Argonaute family members have been shown to be involved in RNAi in Neurospora crassa (QDE3) as well as in A. thaliana (AGO1) (Fagard, M., S. Boutet, J.-B. Morel, C. Bellini, and H. Vaucheret. 2000). Recently, two independent groups identified additional components of the RISC complex. Hammond and group showed the presence of two RNA binding proteins, the Vasa intronic gene and dFMR proteins, in the RISC complex isolated from Drosophila flies (Caudy, A. A., M. Myers, G. J. Hannon, and S. M. Hammond. 2002). Of these, dFMR is a homologue of the human fragile X mental retardation protein. In a parallel study, Siomi and group also isolated a novel ribonucleic protein complex from the Drosophila lysate that contained dFMRI, AGO2, a Drosophila homologue of p68 RNA helicase (Dmp68), and two ribosomal proteins, L5 and L11, along with 5S rRNA (Ischizuka, A., M. C. Siomi, and H. Siomi. 2002). Both of these groups showed not only the presence of these components in the RISC complex, but also interactions among these proteins in vitro. Other components of RISC have not been clearly established yet. Nevertheless, some of the proteins mentioned below could very well constitute the RISC complex.

Figure 1: This showing the general scheme of RNAi functioning, guide sequence is processed through DICER in cytoplasm and based on homology knockdoun or degrade the targated sequence.

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Methods to Induce RNAi in Plants In RNAi research field, one of the biggest challenges is the delivery of the active molecules that will trigger the RNAi pathway in plants. In this system, a number of methods for delivery of dsRNA or siRNA into different cells and tissue include transformation with dsRNA-forming vectors for selected gene(s) by an Agrobacterium mediated transformations (Chuang and Meyerowtiz 2000; Waterhouse et al. 2001); delivery cognate dsRNA of uidA GUS (β-glucuronidase) and TaGLP2a:GFP (green fluorescent protein) reporter genes into single epidermal cells of maize, barley and wheat by particle bombardment (Schweizer et al. 2000), introducing a Tobacco rattle virus (TRV)-based vector in tomato plants by infiltration (Liu et al. 2002a); delivery of dsRNA into tobacco suspension cells by cationic oligopeptide polyarginine-siRNA complex; infecting plants with viral vectors that produce dsRNA (Dalmay et al. 2000a) and delivery of siRNA into cultured plant cells of rice, cotton and slash pine for gene silencing by nanosense pulsed laser-induced stress wave (LISW) (Tang et al. 2006) are being used. Among these the most reliable and commonly used approaches for delivery of dsRNA to plants cells are agroinfiltration, micro-bombardment and VIGS. These are discussed in the following sections. Agroinfiltration Agroinfiltration is a powerful method to study processes connected with RNAi. The injection of Agrobacterium carrying similar DNA constructs into the intracellular spaces of leaves for triggering RNA silencing is known as agroinoculation or agroinfiltration (Hily and Liu 2007). In most cases agroinfiltration is used to initiate systemic silencing or to monitor the effect of suppressor genes. In plants, cytoplasmic RNAi can be induced efficiently by agroinfiltration, similar to a strategy for transient expression of T-DNA vectors after delivery by Agrobacterium tumefaciens. The transiently expressed DNA encodes either an ss- or dsRNA, which is typically a hairpin (hp) RNA. The infiltration of hairpin constructs are especially effective, because their dsRNA can be processed directly to siRNAs, while the constructs expressing ssRNA can also be useful to induce silencing (Johansen and Carrington 2001; Voinnet et al. 2001; Mlotshwa et al. 2002; Tenllado et al. 2003) and for dissecting the mechanism of gene silencing, especially concerned with its suppressors, systemic silencing signal and also for simple protein purification (Johansen and Carrington 2001; Voinnet et al. 2001; Mlotshwa et al. 2002; Tenllado et al. 2003). Besides, they provide a rapid, versatile and convenient way for achieving a very high level of gene expression in a distinct and defined zone.

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Micro-bombardment In this method, a linear or circular template is transferred into the nucleus by micro-bombardment. Synthetic siRNAs are delivered into plants by biolistic pressure to cause silencing of GFP expression. Bombarding cells with particles coated with dsRNA, siRNA or DNA that encode hairpin constructs as well as sense or antisense RNA, activate the RNAi pathway. The silencing effect of RNAi is occasionally detected as early as a day after bombardment, and it continues up to 3 to 4 days post bombardment. Systemic spread of the silencing occurred 2 weeks later to manifest in the vascular tissues of the non-bombarded leaves of Nicotiana benthamiana that were closest to the bombarded ones. After one month or so, the loss of GFP expression was seen in non-vascular tissues as well. RNA blot hybridization with systemic leaves indicated that the biolistically delivered siRNAs induced due to de novo formation of siRNAs, which accumulated to cause systemic silencing (Klahre et al. 2002). Virus induced gene Silencing (VIGS) Modified viruses as RNA silencing triggers are used as a mean for inducing RNA in plants. Different RNA and DNA viruses have been modified to serve as vectors for gene expression (Timmermans et al. 1994; Pogue et al. 2002). Some viruses, such as Tobacco mosaic virus (TMV), Potato virus X (PVX) and TRV, can be used for both protein expression and gene silencing (Kumagai et al. 1995; Angell and Baulcombe 1999; MacFarlane and Popovich 2000; Mallory et al. 2002). All RNA virus-derived expression vectors will not be useful as silencing vectors because many have potent anti-silencing proteins such as TEV (Tobacco etch virus), that directly interfere with host silencing machinery (Kumagai et al. 1995; Palmer and Rybicki 2001). Similarly, DNA viruses have not been used extensively as expression vectors due to their size constraints for movement (Kjemtrup et al. 1998). However, a non- mobile Maize streak Virus (MSV)-derived vector has been successfully used for long- term production of protein in maize cell cultures (Kumagai et al. 1995). Using viral vectors to silence endogenous plant genes requires cloning homologous gene fragments into the virus without compromising viral replication and movement. This was first demonstrated in RNA viruses by inserting sequences into TMV (Dallwitz and Zurcher 1996), and then for DNA viruses by replacing the coat protein gene with a homologous sequence (Kjemtrup et al. 1998). These reports used visible markers for gene silencing phytoene desaturase( PDS) and chalcone synthase (CHS), providing a measure of the tissue specificity of silencing as these have been involved in carotenoid metabolic pathway. The PDS gene acts on the antenna complex of the thylakoid membranes, and protects the chlorophyll from photooxidation. By silencing this gene, a drastic decrease in leaf carotene content resulted into the appearance of photobleaching symptom (Liu et al. 2002; Turnage et al. 2002). Similarly, over

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 125 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 expression of CHS gene, causing an albino phenotype, instead of producing the anticipated deep orange color (Cogoni et al. 1994). As a result, their action as a phenotypic marker helps in easy understanding of the mechanism of gene silencing. Most viruses are plus-strand RNA viruses or satellites, whereas Tomato golden mosaic virus (TGMV) and Cabbage leaf curl virus (CaLCuV) are DNA viruses. Though RNA viruses replicate in the cytoplasm DNA viruses replicate in plant nuclei using the host DNA replication machinery. Both types of viruses induce diffusible, homology-dependent systemic silencing of endogenous genes. However, the extent of silencing spread and the severity of viral symptoms can vary significantly in different host plants and host/virus combinations. With the variety of viruses and the diversity of infection patterns, transmission vectors, and plant defenses it is not surprising that viruses differ with respect to silencing (Teycheney and Tepfer 2001). Because the continuing development of virus-based silencing vectors can extend VIGS to economically important plants, it is useful to consider some of the characteristics of successful VIGS vectors. RNAi in Context to Host-Pathogen System The evolutionary story of RNAi began in the early 1990s with the attempts of Napoli and colleagues who tried to deepen the purple colour by introducing a chalcone synthase gene in Petunia under a strong promoter. Contrary to expectation, the pigmentation in the flowers of transformed plants was not enhanced. Instead, the flowers were de-pigmented and endogenous gene mRNA transcript levels were greatly reduced (Napoli et al. 1990). Because both the transgene and the endogenous gene were suppressed, the observed phenomenon was termed “co-suppression”. Though the mechanistic aspect of this phenomenon remained unknown at that time, post transcriptional gene silencing (PTGS) was not the most accepted proposal (Napoli et al. 1990; Jorgensen et al. 1996; Cogoni and Macino 2000). This phenomenon of suppression of an endogenous gene by transformation with homologous sequences was also observed in the fungus Neurospora crassa where it was termed quelling (Romano and Macino 1992). However, the significance of these observations went unnoticed for several years until the mystery was solved in 1998, when it was demonstrated that dsRNA is even more effective in silencing gene expression than ss antisense RNA, a phenomenon that was termed as RNAi (Fire et al. 1998). Although such gene silencing can occur at the transcriptional level, it was recognized that a major mechanism of gene suppression occurs post transcriptionally and that a major mechanism of this PTGS is RNAi, the selective degradation of mRNAs targeted by siRNA (van Blokland et al. 1994). This mechanism was later on developed as a VIGS system based on sequence homology studies between a virus and either a transgene or an endogenous gene that would cause PTGS (Lindbo et al.

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1993; Kumagai et al. 1995). In this system, a virus vector carrying a copy of the gene to be silenced is introduced into the cell, the cellular machinery recognizes the viral threat and releases a protective defense to destroy not only viral genes but also any extra-gene being carried by the viral vector, affecting any native or transgenic homologous transcripts (Ruiz et al. 1998; Waterhouse et al. 2001). Such PTGS via RNAi can occur rapidly with proteins for many genes, being decreased within hours, and completely absent within 24 h. Based upon these and other findings initially made in studies of plants, it seems very likely that RNAi evolved as a mechanism to defend plant cells against fungal, bacterial, viral and nematode infection (Mann et al. 2008, Wani et al. 2010). RNAi Strategies in Plant Disease Management Despite substantial advances in plant disease management strategies, our global food supply is still threatened by a multitude of pathogens and pests. This changed scenario warrants us to respond more efficiently and effectively to this problem. The situation demands judicious blending of conventional, unconventional and frontier technologies. In this sense, RNAi technology has emerged as one of the most potential and promising strategies for enhancing the building of resistance in plants to combat various fungal, bacterial, viral and nematode diseases causing huge losses in important agricultural crops (Mann et al. 2008, Wani et al. 2010). The nature of this biological phenomenon has been evaluated in a number of host-pathogen systems and effectively used to silence the action of pathogen. Many of the examples listed below illustrate the possibilities for commercial exploitation of this inherent biological mechanism to generate disease-resistant plants in the future by taking advantage of this approach. Management of Plant Pathogenic Fungi RNA-mediated gene silencing (RNA silencing) is used as a reverse tool for gene targeting in fungi. Homology-based gene silencing induced by transgenes (co- suppression), antisense, or dsRNA has been demonstrated in many plant pathogenic fungi, including Cladosporium fulvum (Hamada and Spanu 1998), Magnaporthae oryzae (Kadotani et al. 2003), Venturia inaequalis (Fitzgerald et al. 2004), Neurospora crassa (Goldoni et al. 2004), Aspergillus nidulans (Hammond and Keller 2005), and Fusarium graminearum (Nakayashiki 2005), whether it is suitable for large-scale mutagenesis in fungal pathogens remains to be tested. Hypermorphic mechanism of RNA interference implies that this technique can also be applicable to all those plant pathogenic fungi, which are polyploid and polykaryotic in nature. And also offers a solution to the problem where frequent lack of multiple marker genes in fungi is experienced. Simultaneous silencing of several unrelated genes by

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 127 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 introducing a single chimeric construct has been demonstrated in case of Venturia inaequalis (Fitzgerald et al. 2004). HCf-1, a gene that codes for a hydrophobin of the tomato pathogen C. fulvum (Spanu 1997), was co-suppressed by ectopic integration of homologous transgenes. Transformation of Cladosporium fulvum with DNA containing a truncated copy of the hydrophobin gene HCf-1 caused co-suppression of hydrophobin synthesis in 30% of the transformants. The co-suppressed isolates had a hydrophilic phenotype, lower levels of HCf-1 mRNA than wild type and contain multiple copies of the plasmid integrated as tandem repeats at ectopic sites in the genome (Hamada and Spanu 1998). The transcription rate of HCf-1 in the co-suppressed isolates was higher than in the untransformed strains, suggested that silencing acted at the post-transcriptional level (Hamada and Spanu 1998). This was due to ectopic integration of the transgene next to promoters which initiate transcription to form antisense RNA and that this in turn determines down-regulation of HCf-1. But gene silencing was not associated with DNA cytosine methylation (Hamada and Spanu 1998). Similarly, the silencing of cgl1 and cgl2 genes using the cgl2 hairpin construct in Cladosporium fulvum has also been reported (Segers et al. 1999), though the effect was possibly restricted to highly homologous genes (exons of cgl 1 and cgl 2 are 87% identical). However, the less homologous cgl 3 (53% overall identity to cgl 2) was not affected as the target specificity always depends upon the actual sequence alignment and more over, short regions of high density that led to unwanted off-targets effects. Such a strategy could be exploited for protecting the consumable products of vegetables and fruits crops from the post harvest diseases caused by different plant pathogens in future. Fitzgerald and colleagues (2004), using hairpin vector technology have been able to trigger simultaneous high frequency silencing of a green fluorescent protein (GFP) transgene and an endogenous trihydroxynaphthalene reductase gene (THN) in V. inaequalis. GFP transgene, acting as easily detectable visible marker while the trihydroxynaphthalene reductase gene (THN) playing role in melanin biosynthesis. High frequency gene silencing was achieved using hairpin constructs for the GFP or the THN genes transferred by Agrobacterium (71 and 61%, respectively). THN- silenced transformants exhibited a distinctive light brown phenotype and maintained the ability to infect apple. Silencing of both genes with this construct occurred at a frequency of 51% of all the transformants. All 125 colonies silenced for the GFP gene were also silenced for THN (Fitzgerald et al. 2004). Similarly, multiple gene silencing has been achieved in Cryptococcus neoformans using chimeric hairpin constructs (Liu et al. 2002) and in plants using partial sense constructs (Abbott et al. 2002). The first effort towards the systematic silencing of Magnaporthe grisea, a causal organism of rice blast was carried out in by Kadotani et al. (2003) by using the

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 128 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 enhanced green florescent protein gene as a model. To assess the ability of RNA species to induce silencing in fungus, plasmid construct expressing sense, antisense and hairpin RNA were introduced into an eGFP-expressing transformants. The fluorescence of eGFP in the transformants was silenced much more efficiently by hairpin RNA of eGFP than by other RNA species. In the silenced transformants, the accumulation of eGFP mRNA was drastically reduced. But not methylation of coding or promoter region was involved. The small interfering RNA molecules of 19-23 nucleotides were observed in both sense and antisense strands of eGFP gene (Kadotani et al 2003). Later on Nakayashiki and colleagues (2005) developed a protocol for silencing the mpg1 and polyketide synthase-like genes. mpg1 gene is a hydrophobin gene which is essential for pathogenicity as it act as a cellular relay for adhesion and trigger for the development of appressorium (Talbot et al. 1996). Their work on this host-pathogen system revealed that they were successfully able to silence the above mentioned genes at varying degrees by pSilent-1-based vectors in 70–90% of the resulting transformants. Ten to fifteen percent of the silenced transformants exhibited almost ‘‘null phenotype’’. This vector was also efficiently applicable to silence a GFP reporter in another ascomycete fungus Colletotrichum lagenarium (Nakayashiki 2005). Rust fungi are devastating plant pathogens and several Puccinia species have a large economic impact on wheat production worldwide. Disease protection, mostly offered by introgressed host-resistance genes, is often race-specific and rapidly overcome by newly-emerging virulent strains. Extensive new genomic resources have identified vital pathogenicity genes but their study is hampered because of the biotrophic life styles of rust fungi. In cereals, Barley stripe mosaic virus (BSMV)- induced RNAi has emerged as a useful tool to study loss-of-function phenotypes of candidate genes. Expression of pathogen-derived gene fragments in this system can be used to obtain in planta-generated silencing of corresponding genes inside biotrophic pathogens, a technique termed host-induced gene silencing (HIGS). In view of testing the effectiveness of BSMV-mediated HIGS in the wheat leaf rust fungus Puccinia triticina (Pt) by targeting three predicted pathogenicity genes, a MAPK, a cyclophilin, and a calcineurin regulatory subunit. Inoculation of BSMV RNAi constructs generated fungal gene-specific siRNA molecules in systemic leaves of wheat plant. Subsequent Pt inoculation resulted in a suppressed disease phenotype and a reduction in endogenous transcript levels of the targeted fungal genes indicating translocation of siRNA molecules from host to fungal cells. Efficiency of this host-generated trans- specific RNAi was enhanced by using BSMV silencing vectors defective in coat protein coupled with introducing fungal gene sequences simultaneously in sense and antisense orientation. The disease suppression indicated the likely involvement of these fungal genes in pathogenicity. This demonstrates that BSMV-mediated in

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 129 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 planta-generated RNAi is an effective strategy for functional genomics in rust fungi.(Panwar, McCallum, & Bakkeren, 2013) Management of Plant Pathogenic Bacteria One of the striking examples of bacterial disease management where RNAi showed a remarkable type of gene regulation was documented by Escobar et al. (2001). They developed a crown gall disease management strategy that targets the process of tumourogensis (gall formation) by initiating RNAi of the iaaM and ipt oncogenes. Expression of these genes is a prerequisite for wild type tumor formation. Transgenic Arabidopsis thaliana and Lycopersicon esculentum transformed with RNAi constructs, targeting iaaM and ipt gene(s) showed resistance to crown gall disease. Transgenic plants generated through this technology contained a modified version of these two bacterial gene(s) required to cause the disease and was the first report to manage a major bacterial disease through RNAi. The extra genes recognize and effectively shut down the expression of the corresponding bacterial gene during infection, thus preventing the spread of infection. The incoming bacteria could not make the hormones needed to cause tumors and plants deficient in silencing were hyper-susceptible to A tumefaciens (Dunoyer et al. 2007). Successful infection relied on a potent anti-silencing state established in tumors whereby siRNA synthesis is specifically inhibited. The procedure can be exploited to develop broad-spectrum resistance in ornamental and horticultural plants which are susceptible to crown gall tumorigenesis. This approach can be advocated for the effective management of those pathogens which multiply very rapid and results in tumor formation such as Albugo candida, Synchytrium endobioticum, Erwinia amylovora etc. The natsiRNA (nat- siRNAATGB2) was strongly induced in Arabidopsis upon infection by Pseudomonas syringae pv tomato and down-regulates a PPRL gene that encodes a negative regulator of the RPS2 disease resistance pathway. As a result, the induction of nat- siRNAATGB2 increases the RPS2-mediated race-specific resistance against P. syringae pv tomato in Arabidopsis (Katiyar-Agarwal et al. 2007). Recently, the accumulation of a new class of sRNA, 30 to 40 nucleotides in length, termed long- siRNAs (lsiRNAs), was found associated with P. syringae infection. One of these lsiRNAs, AtlsiRNA-1, contributes to plant bacterial resistance by silencing AtRAP, a negative regulator of plant defense (Katiyar- Agarwal et al. 2007). A Pseudomonas bacterial flagellin derived peptide is found to induce the accumulation of miR393 in Arabidopsis. miR393 negatively regulates mRNAs of F-box auxin receptors, resulting in increased resistance to the bacterium (P. syringae), and the overexpression of miR393 was shown to reduce the plant’s bacterial titer by 5-fold (Navarro et al. 2006).

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The cuticle plays an important role in plant interactions with pathogens and with their surroundings. The cuticle acts as both a physical barrier against physical stresses and pathogens and a chemical deterrent and activator of the plant defense response. Cuticle production in tomato plants is regulated by several transcription factors, including SlSHINE3, an ortholog of the Arabidopsis WIN/SHN3. Here we used a SlSHINE3-overexpressing (SlSHN3-OE) and silenced (Slshn3-RNAi) lines and a mutant in SlCYP86A69 (Slcyp86A69)-a direct target of SlSHN3-to analyze the roles of the leaf cuticle and cutin content and composition in the tomato plant's defense response to the necrotrophic foliar pathogen Botrytis cinerea and the biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria. SlSHN3, which is predominantly expressed in tomato fruit epidermis, also affects tomato leaf cuticle, as morphological alterations in the SlSHN3-OE leaf tissue resulted in shiny, stunted and permeable leaves. SlSHN3-OE leaves accumulated 38 % more cutin monomers than wild-type leaves, while Slshn3-RNAi and Slcyp86A69 plants showed a 40 and 70 % decrease in leaf cutin monomers, respectively. Overexpression of SlSHN3 resulted in resistance to B. cinerea infection and to X. campestris pv. vesicatoria, correlated with cuticle permeability and elevated expression of pathogenesis-related genes PR1a and AOS. Further analysis revealed that B. cinerea- infected Slshn3-RNAi plants are more sensitive to B. cinerea and produce more hydrogen peroxide than wild-type plants. Cutin monomer content and composition differed between SlSHN3-OE, Slcyp86A69, Slshn3-RNAi and wild-type plants, and cutin monomer extracted from SlSHN3-OE plants altered the expression of pathogenesis-related genes in wild-type plants.(Buxdorf et al., 2013) Management of Plant Pathogenic Viruses Antiviral RNAi technology has been used for viral disease management in human cell lines (Bitko and Barik 2001; Gitlin et al. 2002; Jacque et al. 2002; Novina et al. 2002). Such silencing mechanisms (RNAi) can also be exploited to protect and manage viral infections in plants (Waterhouse et al. 2001; Ullu et al. 2002). The effectiveness of the technology in generating virus resistant plants was first reported to PVY in potato, harboring vectors for simultaneous expression of both sense and antisense transcripts of the helper-component proteinase (HC-Pro) gene (Waterhouse et al. 1998). The P1/HC-Pro suppressors from the poty virus inhabited silencing at a step down stream of dsRNA processing, possibly by preventing the unwinding of duplex siRNAs, or the incorporation into RISC or both (Chapman et al. 2004). The utilization of RNAi technology has resulted in inducing immunity reaction against several other viruses in different plant-virus systems (Wani and Sanghera 2010). In phyto -pathogenic DNA viruses like Gemini viruses non-coding intergenic region of Mungbean yellow mosaic India virus (MYMIV) was expressed as hairpin

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 131 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 construct under the control of the 35S promoter and used as biolistically to inoculate MYMIV-infected black gram plants and showed a complete recovery from infection, which lasted until senescence (Pooggin et al. 2003). RNAi mediated silencing of geminiviruses using transient protoplast assay where protoplasts were co-transferred with a siRNA designed to replicase (Rep)-coding sequence of African cassava mosaic virus (ACMV) and the genomic DNA of ACMV resulted in 99% reduction in Rep transcripts and 66% reduction in viral DNA (Vanitharani et al. 2003). It was observed that siRNA was able to silence a closely related strain of ACMV but not a more distantly related virus. About more than 40 viral suppressors have been identified in plant viruses (Ruiz and Voinnet 2007). Results from some of the well-studied virus suppressors indicated that suppressors interfere with systemic signaling for silencing (Mlotshwas et al. 2002). During last few years, the p69 encoded by Turnip yellow mosaic virus has been identified as silencing suppressors that prevented host RDR-dependent secondary dsRNA synthesis (Chen et al. 2004). P14 protein encoded by aureus viruses suppressed both virus and transgene-induced silencing by sequestering both long dsRNA and siRNA without size specificity (Merai et al. 2005). Multiple suppressors have been reported in Citrus tristeza virus where p20 and coat protein (CP) play important role in suppression of silencing signal and p23 inhibited intracellular silencing (Lu et al. 2004). Multiple viral components, viral RNAs and putative RNA replicase proteins were reported for a silencing or suppression of Red clover necrotic mosaic virus (Takeda et al. 2005). In this case, the RNA silencing machinery deprived of DICER-like enzymes by the viral replication complexes appears to be the cause of the suppression. Pns10 encoded by Rice dwarf virus suppressed local and systemic S-PTGS but not IR-PTGS suggesting that Pns10 also targets an upstream step of dsRNA formation in the silencing pathway (Cao et al. 2005). Niu and colleagues (2006) used a 273-bp (base pair) sequence of the Arabidopsis miR159 a pre-miRNA transcript expressing amiRNAs against the viral suppressor genes P69 and HC-Pro to provide resistance against Turnip yellow mosaic virus and Turnip mosaic virus infection, respectively. In addition, a dimeric construct harboring two unique amiRNAs against both viral suppressors conferred resistance against these two viruses in inoculated Arabidopsis plants. Similarly, Qu et al. (2007) used a different amiRNA vector to target the 2 b viral suppressor of the Cucumber mosaic virus (CMV), a suppressor that interacted with and blocked the slicer activity of AGO1 had also shown to confer resistance to CMV infection in transgenic tobacco. A strong correlation between virus resistance and the expression level of the 2 b-specific amiRNA was shown for individual plant lines.

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One of the major roles of RNAi in plants and invertebrates is antiviral defense. Conversely, viruses have also evolved to encode suppressors of RNAi (VSRs), which disrupt RNAi at various steps. Research activities focusing on the relationship between plant viruses and RNAi have been essential to our current understanding of RNAi mechanisms.(Qu, n.d.) It is evident from above-mentioned reports that the RNA components, such as single strand template RNA, dsRNA and/or siRNA of the silencing pathways are the preferred targets of most viral suppressors. However, plant viruses are known to have evolved a counter-silencing mechanism by encoding proteins that can overcome such resistance (Li and Ding 2006; Díaz-Pendón and Ding 2008). These suppressors of gene silencing are often involved in viral pathogenicity, mediate synergism among plant viruses and result in the induction of more severe disease. Simultaneous silencing of such diverse plant viruses can be achieved by designing hairpin structures that can target a distinct virus in a single construct (Díaz-Pendón and Ding 2008). Contrarily, the RNAi system may cause an increase in the severity of viral pathogenesis and/or encode proteins, which can inactivate essential genes in the RNAi machinery (Elbashir et al. 2001; Li et al. 2002) that helps them in their replication in the host genome (Hannon 2002). Management of Plant Parasitic Nematodes Several major plant parasitic nematodes such as the root-knot (Meloidogyne spp.) and cyst (Heterodera spp.) along with other minor nematodes cause significant damage to important crops like legumes, vegetables and cereals in most parts of the world and continue to threaten these agricultural crops. So a natural, eco-friendly defense strategy that delivers a cost-effective control of plant parasitic nematodes is needed which is difficult to achieve through conventional approaches. However, the birth of RNAi technology from classical C. elegans studies has shown the ways and means to explore the possibilities of this mechanism for protecting plants from nematode damage. In this context, two approaches have been advocated, one of them relies on targeting plant genes that are involved with the infection process, and the second approach targets essential genes within the nematode. RNAi can be induced in C. elegans by feeding it dsRNA, so it was reasoned that expressing hpRNAs containing sequences of vital nematode genes in the host plant might deliver dsRNA to a feeding nematode to incapacitate or kill it. After the demonstration of gene silencing using siRNA duplexes in the nematode (Fire et al. 1998), the use of RNAi has rapidly emerged as the technique of choice for plant nematologists to put their efforts, especially for nematode management in agriculture. RNAi-mediated suppression of a gene plays an indispensable role in hampering the nematode development and may adversely affect

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 133 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 the progression of pathogenesis in direct or indirect ways. There are accumulating evidences for the efficacy of RNAi in plant parasitic nematode management and a wide range of genes have been targeted for silencing in cyst and root-knot nematode species (Mann et al. 2008, Sanghera et al. 2010). RNAi in the context of phyto-parasitic nematodes was used as early as the beginning of this century, when stimulation of oral ingestion by second-stage juveniles of cyst nematodes H. glycines, G. pallida (Urwin et al. 2002) and root-knot nematode M. incognita (Bakhetia et al. 2005) was achieved by using octopamine. Later on, resorcinol- and serotonin-inducing dsRNA uptake by second stage juvenile of M. incognita was found to be more effective than octopimine (Rosso et al. 2005). The genes targeted by RNAi to date are expressed in a range of different tissues and cell types. The ingested dsRNA can silence genes in the intestine (Urwin et al. 2002; Shingles et al. 2007), female reproductive system (Lilley et al. 2005), sperm (Urwin et al. 2002; Steeves et al. 2006), and both subventral and dorsal oesophageal glands (Chen et al. 2005; Rosso et al. 2005; Huang et al. 2006; Bakhetia et al. 2007). Uptake of dsRNA from the gut is a proven route to systemic RNAi in C. elegans. The systemic nature of RNAi in plant parasitic nematodes following ingestion of dsRNA suggests that they share similar uptake and dispersal pathways. However, RNAi of a chitin synthase gene expressed in the eggs of Meloidogyne artiella was achieved by soaking intact eggs contained within their gelatinous matrix in a solution containing dsRNA (Fanelli et al. 2005). The enzyme plays a role in the synthesis of the chitinous layer in the eggshell. Depletion of its transcript by RNAi led to a reduction in stainable chitin in eggshells and a delay in hatching of juveniles from treated eggs. Similarly, RNAi targeting for cysteine proteinase transcripts did not reduce parasitic population of established nematodes on plants but result into the alteration of their sexual fate in favour of males at 14 days after invasion (Urwin et al. 2002). On the other hand H. glycines exposed to dsRNA corresponding to a protein with homology to C-type lectins did not affect sexual fate, but 41% fewer nematodes were recovered from the plants (Urwin et al. 2002). But treatment with dsRNA corresponding to the major sperm protein (MSP) had no effect on nematode development or sexual fate 14 days after treatment. In addition to this, reduction in transcript abundance for targeted mRNAs in the infective juvenile and for MSP transcripts when males reached sexual maturity and sperm are produced was observed (Urwin et al. 2002). In further extension of such types of experiments showed efficient FITC uptake by soaking M. incognita, 90-95% of individuals swallowed the dye when the target was a dual oxidase (an enzyme comprised with a peroxidase domain EF-hands and NADPH oxidase domain and potentially involved in

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 134 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170 extracellular matrix development). The effect of RNAi was observed when root knot nematode (RKN) juveniles were fed on dual oxidase-derived dsRNA, the reduction in the number and size of established females at 14 and 35 days post infection with an overall reduction of 70% in egg production was observed (Bakhetia et al. 2005). RNAi has also been induced for a chitin synthase gene that is expressed in the eggshells of M. artiella after soaking its developing eggs in a dsRNA (Fanelli et al. 2005). Heterodera schachtii induces syncytial feeding structures in the roots of host plants, and this requires the up-regulation of Suc transporter genes to facilitate increased nutrient flow to the developing structure. Targeting these genes and down-regulating them with RNA silencing resulted in a significant reduction of female nematode development (Hoffman et al. 2008). Indeed, tobacco plants transformed with hpRNA constructs against two such root-knot nematode genes have shown such an effect: the target mRNAs in the plant parasitic nematodes were dramatically reduced, and the plants showed effective resistance against the parasite (Fairbairn et al. 2007).

The Hemipteran insect brown planthopper (Nilaparvata lugens Sta ˚l) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, genes identified (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced. This shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. (Zha et al., 2011) The pest resistance is improved in transgenic tobacco plants expressing dsRNA of EcR from the cotton bollworm, Helicoverpa armigera, a serious lepidopteran pest for a variety of crops. When H. armigera larvae were fed with the whole transgenic tobacco plants expressing EcR dsRNA, resistance to H. armigera was significantly improved in transgenic plants. Meanwhile, when H. armigera larvae were fed with leaves of transgenic tobacco plants expressing EcR dsRNA, its EcR mRNA level was dramatically decreased causing molting defects and larval lethality. In addition, the transgenic tobacco plants expressing H. armigera EcR dsRNA were also resistant to another lepidopteran pest, the beet armyworm, Spodoptera exigua, due to the high similarity in the nucleotide sequences of their EcR genes. This study provides additional evidence that transgenic plant expressing dsRNA targeting insect- associated genes is able to improve pest resistance.(Zhu et al., 2012)

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 135 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

The gap gene hunchback (hb) is of crucial importance in insect axial patterning and knockdown of hb is deforming and lethal to the next generation. The peach potato aphid, Myzus persicae (Sulzer), has many host plants and can be found throughout the world. To investigate the effect of plant-mediated RNAi on control of this insect, the hb gene in M. persicae was cloned, plant RNAi vector was constructed, and transgenic tobacco expressing Mphb dsRNA was developed. Transgenic tobacco had a different integration pattern of the transgene. Bioassays revealed that continuous feeding of transgenic diet reduced Mphb mRNA level in the fed aphids and inhibited insect reproduction, indicating successful knockdown of the target gene in M. persicae by plant-mediated RNAi.(Mao & Zeng, 2013) Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is pest of plants, providing potential novel opportunities for insect pest control. The putative Snf7 homolog of WCR (DvSnf7) has shown to be an effective RNAi target for insect control, as DvSnf7 RNAi leads to lethality of WCR larvae. Snf7 functions as a part of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway which plays a crucial role in cellular housekeeping by internalization, transport, sorting and lysosomal degradation of transmembrane proteins. Study indicates that ubiquitinated proteins accumulate in DvSnf7 dsRNA-fed larval tissues and that the autophagy process seems to be impaired. These findings suggest that the malfunctioning of these cellular processes in both midgut and fat body tissues triggered by DvSnf7 RNAi were the main effects leading to the death of WCR.(Ramaseshadri et al., 2013) Management of plant stress In plant many stress specific genes are expressed in response to various biotic and abiotic factors, by the application of RNAi the downregulation and their knockdown can be done very specifically and plants can be made stress resistance. For example Arabidopsis RING E3 AtAIRP3/LOG2 is a positive regulator of the ABA-mediated drought and salt stress tolerance mechanism. Using yeast (Saccharomyces cerevisiae) two-hybrid, in vitro, and in vivo immunoprecipitation, cell-free protein degradation, and in vitro ubiquitination assays, RESPONSIVE TO DEHYDRATION21 was identified as a substrate protein of AtAIRP3/LOG2. AtAIRP3/LOG2 plays dual functions in ABA-mediated drought stress responses and in an amino acid export pathway in Arabidopsis. A New Gene (RING) E3 ubiquitin ligases have been implicated in cellular responses to the stress hormone abscisic acid (ABA) as well as to environmental stresses in higher plants. ABA-insensitive RING protein3 (atairp3) loss of-function mutant line in Arabidopsis (Arabidopsis thaliana) was isolated due to its hyposensitivity to ABA during its germination stage as compared with wild-type plants. AtAIRP3 contains a single C3HC4-type RING motif, a putative myristoylation site, and a domain associated with RING2 (DAR2) domain.

Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P.) 470 003 India 136 Journal of the Botanical Society, University of Saugor, Vol. 44, 2014; ISSN 2229-7170

Unexpectedly, AtAIRP3 was identified as LOSS OF GDU2 (LOG2), which was recently shown to participate in an amino acid export system via interaction with GLUTAMINE DUMPER1. Thus, AtAIRP3 was renamed as AtAIRP3/LOG2. Transcript levels of AtAIRP3/LOG2 were up-regulated by drought, high salinity, and ABA, suggesting a role for this factor in abiotic stress responses. The atairp3/log2-2 knockout mutant and 35S:AtAIRP3-RNAi knockdown transgenic plants displayed impaired ABA-mediated seed germination and stomata closure. Cosuppression and complementation studies further supported a positive role for AtAIRP3/LOG2 in ABA responses. Suppression of AtAIRP3/LOG2 resulted in marked hypersensitive phenotypes toward high salinity and water deficit relative to wild-type plants. (Kim & Kim, 2013) Conclusions The field of RNAi is moving at an impressive pace and generating exciting results associated with RNAi, transgene silencing and transposon mobilization. This technology can be considered an eco-friendly, biosafe and ever green technology as it eliminates even certain risks associated with development of transgenic plants carrying first generation constructs (binary vectors and sense and antisense genes). As witnessed from earlier strategies for obtaining viral resistant plants, the expression of protein product from the transgene of interest risked hetero-encapsidation through protein-protein interactions between target and non-target viral gene product, resulted in the development of a non-aphid transmissible strain of Zucchini yellow mosaic virus to aphid-transmissible strain from a transgene expressing a plum pox capsid protein (Lecoq et al. 1993). Since RNAi triggers the formation of dsRNA molecules that target and facilitate the degradation of the gene of interest as well as the transgene itself to avoid problems arising from the synthesis of gene sequences as well as non- coding regions of gene, thus limiting undesirable recombination events. Keeping in view the potentialities of RNAi technology and lesson from this classical example demonstrated that why and how this technology has emerged to combat plant pathogens in the near future as it has already added new dimensions in the chapter of plant disease management. However, a better and comprehensive understanding of RNAi should allow future plant breeders and pathologists to work effectively and efficiently.

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