Advances in Bioresearch ORIGINAL ARTICLE Influence of AM fungi ( Glomus mosseae, Acaulospora laevis and Gigaspora

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1 Advances in Bioresearch Adv. Biores., Vol4 (2) June 2013: Society of Education, India Print ISSN ; Online ISSN Journal s URL: CODEN: ABRDC3 A B R ORIGINAL ARTICLE Influence of AM fungi (Glomus mosseae, Acaulospora laevis and sp.) alone and in Combination with Trichoderma viride on Growth Responses and Physiological Parameters of Dianthus caryophyllus Linn. Sayeeda Kousar Bhatti*, Aditya Kumar**, Tanuja Rana & Navroop Kaur School of Biological Sciences, Shoolini University, Solan, Himachal Pradesh , INDIA **Department of Botany, Dayanand Post Graduate College, Hisar sayeedakhan6@gmail.com ABSTRACT Mycorrhiza is an apparent structure developed as a result of symbiotic association between fungi and plant roots, whereby the plant provides sugar for the fungi and the fungi give nutrients such as phosphorus, water and protection to the plants. In the present research work, three AM fungi (Glomus mosseae, Acaulospora laevis and sp.) along with T. viride alone or in different combinations were evaluated for their efficacy in improving the growth performance of Dianthus caryophyllus Linn. After 45 and 90 days of inoculation all the inoculated plants showed better growth responses over control plants. The maximum mycorrhizal effect on experimental plants was observed by the dual inoculation of G.mosseae and T.viride. KEYWORDS: Symbiotic association, Arbascular mycorrhizal fungi, Glomus mosseae Acaulospora laevis, sp., Trichoderma viride, Dianthus caryophyllus, Inoculation Received: 14/04/2013 Accepted: 08/05/ Society of Education, India INTRODUCTION The term mycorrhiza was coined by A.B. Frank in 1885 [1]. Mycorrhiza is the mutualistic symbiotic association (non-pathogenic) between soils borne fungi with the roots of higher plants [2]. More than 6000 fungal species are capable of establishing mycorrhizal association of approximately with 2, 40,000 plant species [3]. Mycorrhizal symbiosis is an important natural phenomenon of plant and soil fungi interaction. An arbuscular mycorrhiza is a type of mycorrhiza in which the fungus penetrates the cortical cells of the roots of a vascular plant. AM fungi are symbiotic associations formed between the members of phylum Glomeromycota and the roots of most terrestrial flowering plants. Arbuscular mycorrhizal fungi (AMF) are globally distributed soil microorganisms that form symbiotic associations with more than 80% of terrestrial plants [4]. The role of AM fungi is well known. They provide important nutrient absorption system for mineral nutrients like nitrogen, phosphorus, potassium and trace elements like zinc, manganese etc. to the plant and also make available soluble form of phosphate. They improve disease resistance, drought resistance and survival of host plant under stress conditions and being used to tolerate transplantation shocks in micro-propagated plants [5]. Many new modern techniques, methods, strategies and plant associations have been put into use to improve the quantity and quality of plant resources. In ornamental flowering plants AM fungi is consider as the source of biofertilizers. AM fungi are also responsible to increase the plant height, root and shoot biomass, chlorophyll content, phosphorus content, root length, increase flower production, drought resistance, etc. Ornamental flowering plants have a great significance due to the large demand of flowers hence, cultivation of ornamental plants has received an impetus in the recent years. It becomes necessary to enhance their biomass production and their quality in order to fulfil the need of society. Therefore, it requires formulation of planning and strategies for their conservation and enhancement of their products. However, the outputs are limited because of low level of fertilizers used or hazardous effect of heavy ABR Vol 4 [2] June P a g e 2013 Society of Education, India

2 doses of chemical fertilizers and of the care taken to harvest. Mycorrhizal inoculation can be a suitable way to improve growth and root development of ornamental flowering plants, particularly in poor soils. Therefore, it is advisable to develop cheaper solution as employing mycorrhizal inoculation which has gained momentum in last couple of years. In the present study, the purpose of using an AM fungi (Glomus mosseae, Acaulospora laevis and sp.) alone and in combination with Trichoderma viride was to assess which one is better for acclimatization and for various growth parameters of Dianthus caryophyllus after 45 and 90 days of inoculation. Therefore after 45 days and 90 days of experimentation different parameters were assessed to see the effect on inoculated plants over control plants with different treatments which are mentioned as change in height, root length, shoot weight, root weight, chlorophyll content, phosphorus content, AM spore number and % AM root colonization. The Greek botanist Theophrastus gave it the botanical name "Dianthus" meaning "flower of the gods." Dianthus caryophyllus belongs to the family Caryophyllaceae. It is an annual herb grown in the gardens as ornamental and decorative purposes. They are present at weddings, birthday parties and festivals. In Western herbal medicine the entire plant is used as a bitter tonic herb that stimulates the urinary system and digestive system. MATERIAL AND METHODS AM inoculums: The AM fungal species (Glomus mosseae, Acaulospora laevis and sp.) were mass multiplied by using trap plant (Triticm aestivum) and were used for inoculation. The starter culture was percured from Department of Botany, Kurukshetra University, Kurukshetra and University of Pune, Pune. Mass production of Trichoderma viride: T. viride was isolated from the soil by Warcup s soil plate method and mass cultured by using wheat bran: saw dust medium. Preparation of pot mixture: One month old seedlings of preferred ornamental flowering plants were procured from Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh. Seedlings were grown in earthern pots. To each pot 10% inoculum of each AM fungi and T.viride, alone and in combination was added. Plants were nourished with 150ml. Hoagland nutrient solution after every 10 days during the course of experimentation. Sample collection: Soil samples were collected from the root zone of each plant after 45 days and 90 days. The collected soil samples were wet sieved for isolation of AM spores by using 'Wet Sieving and Decanting Technique' of Gerdemann and Nicolson [6] and quantified by Grid Line Intersect Method by Adholeya and Gaur [7]. After 45 and 90 days of inoculation terminal roots were removed from the root, rinsed with tap water and stained with trypan blue. The AM root colonization was studied according to rapid clearing and staining method of Phillips and Hayman [8]. The percentage of mycorrhizal root colonization was calculated by following formula; No. of root segments infected % age root colonization = 100 Total no. of root segments studied Observation: After 45 days and 90 days of experimentation various parameters were assessed to see the effect on inoculated plants over control plants. The growth parameters of inoculated plants like change in plant height (measured in cm. from soil surface to the growing tip of plant), root length (cm.), shoot weight and root weight (gm.), and the oven dried to 70ºC for dry weight (gm.), chlorophyll content [9], phosphorus content (by vanado-phosphoric acid yellow colour method in nitric acid system outlined by Jackson, 1973), AM spore number and percentage AM root colonization were assessed [10]. Fig 1: Initial height of the plants ABR Vol 4 [2] June P a g e 2013 Society of Education, India

3 Fig 2: Height of the plants after 45 days of inoculation Fig 3: Height of the plants after 90 days of inoculation RESULT AND DISCUSSION Arbuscular mycorrhizal fungi are well known to improve the nutritional status and thereby aid in increased growth and yield of Dianthus caryophyllus. The results of the present study clearly brought out the beneficial effect of inoculation with AM fungi (G.mosseae, A.laevis and sp.) and T.viride on the growth and physiological parameters of Dianthus caryophyllus like plant height, root length, shoot and root biomass, AM spore number, % AM root colonization, chlorophyll and phosphorus contents. Result showed that the plants under investigation varied in their responses to inoculation with AM fungi along with T. viride alone or in different combinations and clearly showed that such type of inoculation improved the growth and yield of Dianthus caryophyllus (Table- 1.1 to 1.4). Shoot length: The data presented in tables (1.1 and 1.3) showed that AM fungi increased shoot length of Dianthus caryophyllus after 45 and 90 days of inoculation. It was observed that all the inoculated plants showed better increase in height over control plants. It was found that change in shoot length was maximum in dual combination of G. mosseae + T.viride (25.83 ± 0.76; 40.44± 0.19). It was found that in a single inoculation, G. mosseae (24.3 ± 0.36; 38.03±0.33) proved to be much better in comparison to other treatments. However, the minimum shoot length was observed in control plants ( ± 0.45; ± 0.33) after inoculation. It was proved that AM inoculation along with T.viride alone or in different combinations significantly improved the shoot length of the plants. Recently it was found that AM fungi were found to have a significant effect on the growth and development of Catharanthus roseus [11]. Inoculation with G. mosseae plus A. laevis significantly increased plant height as compared to uninoculated plants [12]. Shoot Biomass: It is evident from the table (1.1 and 1.3) that forty five and ninty days after inoculation, the maximum increase in shoot biomass (fresh and dry weight) was recorded in plants inoculated with dual combination of G. mosseae plus T.viride (15.06 ± 0.15, 2.73± 0.03; ± 0.77, 7.23 ± 0.23). The shoot biomass increased due to the maximum branched shoots of this plant as compared to other treatments. It is evident that increased nitrogen and phosphorus uptake could also subsequently lead to increased plant height, biomass and leading to increased productivity [13]. Mycorrhizal inoculation significantly increased the biomass, root development (volume, surface area, diameter and number of tips) and photosynthetic pigments of Zizyphus species in comparison to non mycorrhizal plants [14]. Root Biomass: Root Biomass (fresh and dry) of inoculated plants was increased as compared to control plants (Table- 1.1, 1.3). The highest root biomass was observed in the dual combination of sp. plus T. viride after 45 days and 90 days of inoculation (3.33± 0.11, 1.71± 0.07; 6.43 ±0.01, 2.03 ± 0.27). Highly branched roots were found in this dual combination of sp plus T. viride. Mycorrhizal fungi decrease the meristematic activity of root apices and thus lead to an increase in the number of adventitious roots [15]. ABR Vol 4 [2] June P a g e 2013 Society of Education, India

4 Recently, it was found that Lilium longiflorum inoculated with Trichoderma sp. and Glomus sp. showed significant effects on flower stalk, length of flower bud, flower head diameter, fresh shoot weight and fresh root weight [16]. Table- 1: Influence of AM Fungi and T.viride on growth performance of Dianthus caryophyllus after 45 days G.mosseae +A.laevis + sp ± 0.30 cd 10.63±0.32 a 9.73± 0.35 d 1.97± 0.05 c 1.53 ± 0.03 e 0.71± 0.03 d ±1.23 ab ±1.47 ab sp. + T. viride ± 0.02 c 7.56 ± 0.15 d 9.43± 0.15 e 1.72± 0.01 d 3.33± 0.11 a 1.71± 0.07 a 45.33± 2.00 b 55.66± 1.36 a A.laevis + T.viride 17.66± 0.45 d 7.26 ± 0.15 e 10.53± 0.14 c 1.99± 0.05 bc 2.03 ± 0.01 c 1.08± 0.11 bc 57.33± 1.44 a 50.00±0.03 ab G.mosseae +T.viride ± 0.76 a 8.06 ± 0.15 c ± 0.15 a 2.73± 0.03 a 1.16± 0.10 g 0.60± 0.09 de 35.33± 2.32 c ± 2.01 b sp ±0.55 cd 7.13 ± 0.49 f 8.56 ± 0.11 f 1.78± 0.01 d 3.21± 0.00 b 1.67± 0.12 a 43.33±1.46 b ± 0.77 c Acaulospora laevis ± 0.35 d 6.96 ± 0.20 g 7.18 ± 0.03 g 1.30± 0.04 e 2.05 ± 0.05 c 0.99± 0.06 c 55.66±1.05 a ± 0.58 d Glomus mosseae 24.3 ± 0.36 b 9.43 ± 0.30 b ± 0.30 b 2.05± 0.02 b 1.41 ± 0.05 f 0.67± 0.05 d 37.33± 2.24 c 33.00±3.02 de Trichoderm a viride 18.13± 0.32 d 8.03± 0.20 c 6.33 ± 0.20 h 0.99± 0.01 f 1.59 ± 0.03 d 0.87± 0.04 cd 36.66± 3.21 c 31.33± 1.51 e Control ± 0.45 e 4.06± 0.15 h 4.66± 0.35 i 0.45± 0.03 g 1.02 ± 0.06 h 0.29± 0.01 f ± 1.00 d ± 1.27 f Treatments Change in height (cm.) Root length (cm.) Fresh shoot weight (gm.) Dry shoot weight (gm.) Fresh root weight (gm.) Dry root weight (gm.) AM spore number/10 gm. of soil AM root colonization (%) *Each value is an average of three replicates. Mean values followed by different alphabet/s are significant over one another by Duncan s Multiple Range Test at P = ± Standard Deviation. ABR Vol 4 [2] June P a g e 2013 Society of Education, India

5 Table- 1.2: Effect of AM Fungi and T.viride on physiological parameters of Dianthus caryophyllus after 45 days Chlorophyll Content (mg./gm.fresh wt.) % Phosphorus Content Treatments Chl. a Chl. b Total Chlorophyll Shoot P Root P Control ± 0.04 i 0.234± 0.02 a ± 0.06 d 0.278±0.02 g 0.137±0.03 h Trichoderma viride 0.523± 0.02 f 0.348± 0.03 a ± 0.05 c 0.642±0.03 d 0.373±0.01 g Glomus mosseae 1.510± 0.01 d 0.549± 0.01 a ± 0.02 a 0.609±0.05 e 0.537±0.04 b Acaulospora laevis 0.095± 0.02 h 0.421± 0.02 a ± 0.04 d 0.579±0.03 f 0.452±0.02 d sp ± 0.03 e 0.532± 0.01 a ± 0.04 b 0.771±0.01 b 0.369±0.03 g G.mosseae +T.viride 1.947± 0.03 a 0.142± 0.02 a ± 0.05 a 0.647±0.04 d 0.557±0.01 a A.laevis + T.viride 0.356± 0.05 g 0.279± 0.01 a ± 0.06 c 0.678±0.04 c 0.433±0.03 e sp. + T. viride 1.567± 0.01 c 0.454± 0.01 a ± 0.02 a 0.789±0.02 a 0.398±0.01 f G.mosseae +A.laevis + sp ± 0.05 b 0.510± 0.02 a ± 0.07 a 0.584±0.01 f 0.503±0.02 c *Each value is an average of three replicates. Mean values followed by different alphabet/s are significant over one another by Duncan s Multiple Range Test at P = ± Standard Deviation. Table- 1.3: Influence of AM Fungi and T.viride on growth performance of Dianthus caryophyllus after 90 days sp ± 0.45 f 13.33±0.49 b ± 0.21 g 3.98± 0.31 e 5.56± 0.02 b 1.82± 0.12 b 53.00±2.00 d 59.33±0.93 c Acaulospora laevis 28.23± 0.19 h 8.96 ± 0.20 h ± 0.02 b 6.94± 0.44 b 4.75 ± 0.08 d 1.61± 0.74 d 64.33±2.33 b 47.66±1.23 d Glomus mosseae 38.03±0.33 b 12.43±0.30 c ± 0.23 e 4.15± 0.22 e 3.11 ± 0.04 h 1.04± 0.11 h 51.00±2.37 de 37.00±0.33 e Trichoderm a viride 29 33± 0.54 g 11.23±0.21 e ± 0.20 h 2.92± 0.31 f 3.99 ± 0.07 f 1.27± 0.21 f 49.00±3.04 e 39.33±0.15 e Control ± 0.33 i 6.07± 0.25 i 10.66± 0.33 i 1.45± 0.24 g 2.42 ± 0.06 i 0.99± 0.01 h 31.33±1.74 f 27.33±1.26 f Treatments Change in height (cm.) Root length (cm.) Fresh shoot weight (gm.) Dry shoot weight (gm.) Fresh root weight (gm.) Dry root weight (gm.) AM spore number/10 gm. of soil AM root colonizatio n (%) ABR Vol 4 [2] June P a g e 2013 Society of Education, India

6 G.mosseae +A.laevis + sp ±0.23 c 14.02±0.37 a 36.71± 0.35 c 6.07± 0.55 c 3.43±0.06 g 1.14± 0.09 g 52.33±1.23 d 62.33±1.33 b sp. + T. viride 35.08± 0.46 d 9.56± 0.15 g 34.33± 0.19 d 5.72± 0.11 d 6.43 ± 0.01 a 2.03 ± 0.27 a 57.33±2.74 c 69.33±0.19 a A.laevis + T.viride 31.56± 0.09 e 10.26±0.15 f 30.23± 0.24 f 4.01± 0.35 e 4.33 ± 0.00 e 1.48± 0.11 e 76.33±3.74 a 50.66±0.86 d G.mosseae +T.viride 40.44±0.19 a 12.06±0.05 d ± 0.77 a 7.23 ± 0.23 a 4.96± 0.10 c 1.73± 0.33 c 61.33±1.07 b 64.33±2.02 b *Each value is an average of three replicates. Mean values followed by different alphabet/s are significant over one another by Duncan s Multiple Range Test at P = 0.05 ± Standard Deviation. Table- 1.4: Effect of AM Fungi and T.viride on physiological parameters of Dianthus caryophyllus after 90 days Chlorophyll Content (mg./gm.fresh wt.) % Phosphorus Content Treatments Chl. a Chl. b Total Chlorophyll Shoot P Root P Control 0.523± 0.02 h 0.347± 0.03 f 0.870± 0.05 d 0.475±0.03 i 0.337±0.03 h Trichoderma viride 1.012± 0.01 g 0.807± 0.04 a ± 0.05 c 0.982±0.03 h 0.573±0.02 g Glomus mosseae 2.102± 0.01 c 0.812± 0.03 a ± 0.04 b 1.009±0.01 f 0.887±0.03 a Acaulospora laevis 1.099± 0.01 f 0.773± 0.02 d 1.872± 0.03 c 1.179±0.03 b 0.572±0.01 g sp ± 0.03 d 0.799± 0.02 b ± 0.05 b 1.071±0.02 d 0.769±0.03 c G.mosseae +T.viride 2.211± 0.01 b 0.747± 0.04 e 2.958± 0.05 b 1.047±0.03 e 0.817±0.02 b A.laevis + T.viride 1.274± 0.00 e 0.789± 0.02 c 2.063± 0.02 b 0.998±0.04 g 0.733±0.03 d sp. + T. viride 2.012± 0.03 d 0.811± 0.03 a ± 0.06 b 1.109±0.01 c 0.699±0.01 f G.mosseae +A.laevis + sp ± 0.03 a 0.791± 0.01 c ±0.04 a 1.284±0.02 a 0.723±0.02 e Each value is an average of three replicates. Mean values followed by different alphabet/s are significant over one another by Duncan s Multiple Range Test at P = ± Standard Deviation Root length: Inoculation with AM fungi significantly increased the root length of the Dianthus caryophyllus after 45 and 90 days. (Table 1.1, 1.3) indicated that maximum increase in the root length was observed in triple combination of G.mosseae plus A.laevis plus sp (10.63 ± 0.32; 14.02± 0.37). Correlation of root length with mycorrhizal inoculation amount of root is probably related to suitable ventilation of soil that is the result of hypha network of mycorrhizal fungi that connects particles of soil and as result the root spreads into deep soil [17]. Percent AM root colonization and AM spore number: As depicted from results, the percent AM root colonization and AM spore number in soil significantly increased in all the inoculated plants as compared to control plants after 45 and 90 days of inoculation (Table- 1.1, 1.3). Highest percent AM root colonization was found in dual combination of sp. plus T. viride (55.66±1.36; 69.33± 0.19) and also the number of flowers was counted twice and increased flower buds in a single inoculation of sp. Maximum spore number was found in the dual ABR Vol 4 [2] June P a g e 2013 Society of Education, India

7 combination of A.laevis plus T.viride (57.33 ± 1.44; 76.33± 3.74) followed by A.laevis (55.66±1.05; 64.33± 2.33) after inoculation. In the control plants (28.00 ± 1.00; ± 1.74) it was found that AM spore number was minimum. In between the percent AM root colonization and AM spore number there was no positive correlation. Increased AM fungal sporulation and colonization under saltstress conditions has also been reported by Giri and Mukerji (2004) [18]. Mycorrhizal colonization enhanced the growth yield response and a strong correlation was observed between AM colonization and the plant growth [19]. Cholorophyll content: The leaf chlorophyll content recorded in the mycorrhizal plants was typically higher than the control plants. The results in the table (1.2, to 1.4) showed that after 45 and 90 days of inoculation, the maximum increase in chlorophyll a content was recorded in triple combination of G.mosseae plus A.laevis plus sp. (1.612± 0.05; 2.325± 0.03) whereas maximum increase in chlorophyll b content was found in the treatment of G.mosseae (0.549± 0.01; 0.812± 0.03) alone. The maximum amount of total chlorophyll content was found in the plants inoculated with triple combination of G.mosseae plus A.laevis plus sp. (2.122 ± 0.07; ±0.04) followed by G.mosseae plus T.viride (2.089 ± 0.05; 2.958± 0.05) and G. mosseae (2.059 ± 0.02; ± 0.04). In control plants (0.301 ± 0.06; ± 0.05) it was found that total chlorophyll content was minimum. Lotus plants that were inoculated with G. intraradices had higher pure growth, root to stem ratio, sodium to potassium ratio and protein and chlorophyll density than non mycorrhiza inoculated plants [20]. The maximum total chlorophyll content was noticed in G. mosseae inoculated plants followed by those inoculated with G. mosseae and A.laevis. Total chlorophyll content and phosphate content of the shoot was found to be significantly higher in AM inoculated plants as compared to non AM Catharanthus roseus plants. Phosphorus content: Results depicted in Tables (1.2 and 1.4) showed that phosphorus content was significantly increased after 45 and 90 days when plants were subjected to inoculate with AM fungi and T. viride alone or in different combinations. It was found that the amount of phosphorus content in shoots was higher as compared to the roots. After 45 days of inoculation the maximum phosphorus content in shoot and root was found in the dual combination of sp. plus T.viride (0.789±0.02) and G.mosseae plus T.viride (0.557±0.01) respectively. After 90 days of inoculation the maximum increment of phosphorus content in shoot and root was observed in triple combination of G.mosseae plus A.laevis plus sp. (1.284±0.02) and alone G.mosseae (0.887±0.03) respectively. Likewise, the minimum phosphorus content in shoots and roots was observed in control plants (0.278±0.02, 0.475± 0.03; 0.137±0.03, 0.337± 0.03) after 45 and 90 days of inoculation (Table- 1.2, 1.4). Mycorrhizal fungi are well known for their efficient P uptake. Arbuscular mycorrhizal fungi (AMF) are well known for their ability to enhance the phosphorus (P) nutrition of the plants they colonise [21-23]. Soleimanzadeh (2010) studied that head diameter, seed number in head, seed yield and oil yield have been affecting significantly by inoculation with AM fungi because this biofertilizer can enhance absorption of phosphorous by plant [24]. It was found that the vase life of Chrysanthemum sp. flowers increased by inoculation of different bioinoculants as compared to the treated with different growth regulators and nutrients [25]. CONCLUSION The plants under investigation varied in their responses to inoculation with AM fungi and T.viride alone or in different combinations and clearly showed that such type of inoculation improved the growth and yield of Dianthus caryophyllus after 45 and 90 days of inoculation over control. The maximum mycorrhizal effects on Dianthus caryophyllus was observed by the dual inoculation of G.mosseae and T.viride. The conclusion to be drawn from this study is that varied growth responses are expected from inoculation with AM fungi and T.viride on Dianthus caryophyllus. AMF inoculation can differently affect their level of AM root colonization, AM spore number, root and shoot biomass, root length, plant height and also had higher phosphorus and chlorophyll contents than non inoculated plants. As a consequence, AM fungi are currently considered key players in agronomic practices as they may lead to a reduction in the use of chemical fertilizers and pesticides and are therefore potentially important components for the sustainable management of agricultural ecosystems. REFERENCES 1. Frank, A.B. (1885). Über die auf Wurzelsymbiose beruhendc Ernahrung gewisser Baume duren unterirdische Pilzc. Ber.dtsch. Bot. Ges., 3: Sieverding, E. (1991). Vesicular-arbuscular mycorrhiza management intropical agrosystems. Technical Cooperation, Federal Repuplic of Germany Eschborn. ISBN Sharma, M.P., Bhatia, N.P. and Adholya, A. (2001). Mycorrhizal dependency and growth responses of Acacia nitolica and Albizzia lebbeck to inoculation by indigenous AM fungi as influenced by available soil P levels in a semiarid Aflisol wasteland. New For., 21: ABR Vol 4 [2] June P a g e 2013 Society of Education, India

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