EFFECT OF RHIZOBIUM AND PSB IN MYCORRHIZAL LEGUMINOUS PLANTS

Transcription

1 Chapter - 5 EFFECT OF RHIZOBIUM AND PSB IN MYCORRHIZAL LEGUMINOUS PLANTS 5.1 Introduction with review of literature With the introduction of green revolution, the modern agriculture is getting more and more dependent on the steady supply of synthetic inputs i.e. Chemical fertilizers. Indiscriminate and injudicious use of chemical fertilizers for the crop production has compounded the problem of environmental pollution, deterioration of soil health and residue problems. Adverse effects of the chemical fertilizers have compelled the scientific fraternity to look for alternatives in the form of Biofertilizers.The term biofertilizer refers to all organisms, which add, conserve and mobilize the plant nutrients in the soil. Such microorganisms have come to be called as biofertilizers a term, which is a misnomer, compared to commercial fertilizers manufactured on large scales in factories. Biofertilizers are based on renewable energy sources and ecofriendly compared to commercial fertilizers (Yawalkar et al, 1996). Biofertilizers play a very significant role in improving soil fertility by fixing atmospheric nitrogen, both symbiotic and non-symbiotic, solubilise insoluble soil phosphate and produce plant growth substances in the soil. Depending up on the nutrient produced, Verma and Battacharya (Yawalkar et al, 1996) has broadly classified biofertilizers as follows.

2 216 Chapter 5 Biofertilisers Nitrogen fixing Biofertilisers (NBF) Phosphate Mobilizing Biofertilisers NBF for Legumes Rhizobium NBF for Cereals Azospirillum Azotobacter Azolla BGA Phosphate Solubiliser Bacillus Pseudomonas Aspergillus Phosphate Absorber Vesicular Arbuscular Mycorrhiza Biofertilizers can be generally defined as preparations containing live or latent cells of efficient strains of nitrogen fixing, phosphate solubilising or cellulolytic microorganisms, used for application to seed, soil or composting areas with the objective of increasing the number of such microorganisms and accelerate certain microbial processes to augment the extent of the availability of nutrients in the form which can be easily assimilated by plants. Nitrogen is an essential element for plants normal growth and development. Even though nitrogen is present in the atmosphere about 78%, it is not readily available to plants. As nitrogen is diatomic, strong bond is held in between, to break this bond to form nitrogenous compounds, it requires high energy and temperature. But nitrogen-fixing bacteria can break the bond to produce nitrogenous compounds. Two kinds of nitrogen fixing bacterias are there (1) Symbiotic nitrogen fixing bacterias and (2) Asymbiotic (free-living) nitrogen fixing bacterias. In natural environment, the leguminous plants had symbiotic

3 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 217 association of nitrogen fixing bacteria- Rhizobium in the root nodules. Phosphorus is an important plant nutrient, which is referred to as the master key element in crop production (Pierre,1938). The most vital functions of phosphorus in organisms are for cell division, photosynthesis; break down of sugar, energy storage, transfer and as a component of DNA, essential for the passing on of heredity traits from one generation to another. Plants cannot absorb organic phosphorus unless converted to inorganic forms. Certain microbial enzymes could cleave organic phosphorus to inorganic forms available to plants. The enzymes, which cleave phosphorus from organic compounds, are known as phosphatases. Two basic types of phosphatases exist in soils- acid phosphatases which exhibit optimum activity in acidic P H range; and alkaline phosphatases exhibiting optimum activity in the alkaline P H range. The present study is designed to evaluate the effect of Rhizobium and PSB inoculation on mycorrhizal and non-mycorrhizal leguminous plants. 5.2 Materials and Methods Isolation and maintenance of Rhizobium The Nitrogen fixing bacteria were isolated from a naturally growing leguminous plant- Mimosa pudica.l, in (yeast extract maltose agar) YEMA medium (The composition of the medium is given in the appendix) Surface sterilized(using 0.01% mercuric chloride solution) root nodules washed several times in distilled water were crushed in a mortar and pestle. It was serially diluted and at 10 6 dilution pure colony of the rhizobium was obtained.

4 218 Chapter 5 Plate 1 Nitrogen fixing bacteria, Rhizobium leguminosarum L colony developed on YEMA medium. Plate 2 Clearing zone of P solubilising bacteria, Bacillus sp on Apatite Agar medium.

5 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 219 From that pure colony, sub culturing was done. It was maintained in nutrient broth, whose composition is given in the appendix.1ml of the nutrient broth containing about 10 6 bacterial cells, was given to each plant Isolation and maintenance of Phosphate Solubilising Bacteria (PSB) Inoculation of crop plants with phosphate solubilising microorganisms could enhance the utilization of sparingly soluble phosphate by plants. Use of efficient phosphate solubilising microorganisms as seed inoculants or direct use in soil when crops are raised greatly help in phosphate solubilisation and mobilization for crop use Collection of samples-: Soil samples were collected from the rhizosphere of crop plants from 3 different points from a plot. They were mixed and homogenized. 10gms of soil were taken from the mixed samples of soil. They were labeled and stored in polythene bags, at 4 C until they were processed. Pikovskay s medium (1948) was used for the isolation, cultivation and maintenance of Phosphate solubilising Bacteria Enrichment culture technique-: 100ml of pikovskaya s broth sterilized at 10lbs pressure for 30minutes, was dispersed along with 10 gm of rhizosphere soil which was added aseptically in 250ml flask and were incubated at 30 C for one week Phosphatase activity-: The isolated strain of bacteria was cultured in nutrient broth for 72hrs. 1ml of the broth was then transferred to a test tube containing 5ml pre-sterilized basal medium and 5ml of sterilized tris buffer of ph 8.5. To this, 0.5ml of sterilized solution of the substrate, P- nitro phenyl phosphate (0.005m) was added; and 0.5ml of distilled water instead of the substrate served as control. The test tubes were incubated at room temperature for 72hrs. To this added 1ml of 0.2N sodium carbonate and centrifuged at 3000rpm for 15 minutes. The absorbance of supernatant was measured at 418nm. The values of the control tubes were subtracted from the experimental ones for calculating the nitro phenol

6 220 Chapter 5 liberated. The maximum intensity of color developed showed the maximum phosphatase activity. The isolated P solubilising bacteria were grown in nutrient broth in a shaker for 72 hrs. 1ml of the nutrient broth containing 10 6 bacterial cells was given to each plant. Pot culture experiments were conducted to study the effect of coinoculation of PSB and Rhizobium along with VAM fungus in leguminous plants. The soil used in this study was an acidic sandy loam with organic matter- 2%, PH-5.3, total nitrogen- 6.8mg/Kg soil, phosphorus-2.5 mg/kg soil and potassium- 20mg/ Kg soil. The soil was air dried, sieved to less than 2mm and mixed with sand at a ratio of soil: sand 4:1 (v/v) and sterilized at 15 lbs/cm for 30 minutes in an autoclave to eliminate native arbuscular mycorrhizal propagules. The soil was taken in polythene bags, each holding 5 Kg dry soil. The isolation and maintenance of mycorrhizal inoculum of Glomus mosseae was described elsewhere in chapter gm of the soil containing spores and propagules of Glomus. mosseae from the stock culture were laid 1cm below the surface soil. Seeds of four leguminous plants namely Vigna unguiculata and Arachis hypogeae, provided by Kerala Agricultural university farm Kozha, and Agicultural University Tamil Nadu, were grown separately and studied. Seeds were put in water for about 2-4 hrs before sowing. The seeds were surface sterilized in 0.05% mercuric chloride solution and after that thorough washing in glass distilled water. The surface sterilized seeds 3-5 in number were sowed in each bag. 1 ml each of the nutrient broth containing N 2 fixing bacteria- Rhizobium and Phosphate Solubilising Bacteria-Bacillus sp from the stock cultures were also added to the plants as described below

7 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 221 I II III IV V VI VII Treatments Control VAM Rhizobium PSB VAM+ Rhizobium VAM+ PSB VAM+Rhizobium+ PSB The plants were kept in green house with adequate sunlight and ventilation. Daily watering (sterilized and de-ionized water) and observations were done.20ml/pot of modified Hogland s nutrient solution were given once in three days to all the plants under study. The composition of the modified Hogland s solution was given in the appendix. The plants were uprooted after 30, 60 and 120 days of growth. The plants growth rates were assessed by standard parameters like shoot length; shoot weight (fresh & dry), root length, root weight (fresh & dry), nodule number, nodule weight etc. The nutrients NPK uptake and the biochemical constituents like total chlorophyll, total carbohydrates, total reducing sugars and total proteins etc were estimated. Nitrogenase, Acid and alkaline Phosphatases were also studied as per the procedures given in chapter 2.

8 222 Chapter Results Growth rate and nutrient levels Growth rate and NPK levels of Vigna unguiculata and Arachis hypogeae inoculated with Rhizobium, PSB and VAM fungus, after 30, 60 and 120 days of growth were studied and the results are given in tables LCIV to CI. It was found that the co-inoculation of two bacterias with VAM fungus enhanced the growth rate and NPK levels in leguminous plants significantly than the single inoculums and uninoculated plants. Table LCIV: Growth rate of Vigna unguiculata inoculated with Rhizobium, PSB and VAM fungus, after 30 days of growth Shoot length Shoot weight gm cm fresh dry Root Length Root weight (gm) cm fresh dry No. of Nodules Weight of Nodule -1 Gm % of mycorrhizal infection I 20± ± ± ± ± ± II 32± ± ± ± ± ± ±1.45 III 28.3± ± ± ± ± ± ± ± IV 27.6± ± ± ± ± ± V 48.4± ± ± ± ± ± ± ± ±2.31 VI 45.1± ± ± ± ± ± ±1.70 VII 54.3± ± ± ± ± ± ± ± ±2.71 Average of six values in each case ±SD

9 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 223 Table LCV: Growth rate of Vigna unguiculata inoculated with Rhizobium, PSB and VAM fungus, after60 days of growth. Shoot length Shoot weight gm cm fresh dry Root Length cm Root weight (gm) fresh dry No. of Nodules Weight of Nodule -1 gm % of mycorrhizal infection I 28± ± ± ± ± ± II 60± ± ± ± ± ± ±2.74 III 54.3± ± ± ± ± ± ±3 0.04± IV 52.8± ± ± ± ± ± V 78.0± ± ± ± ± ± ±4 0.11± ±3.57 VI 74.7± ± ± ± ± ± ±2.18 VII 86.2± ± ± ± ± ± ± ± ±3.14 Average of six values in each case ± SD

10 224 Chapter 5 Table LCVI: Growth rate of Vigna unguiculata, inoculated with Rhizobium, PSB and VAM fungus after 120 days of growth Shoot length Shoot weight gm cm fresh dry Root Length Root weight (gm) cm fresh dry No. of Nodules Weight of Nodule -1 gm % of mycorrhizal infection I 32.0± ± ± ± ± ± II 88.0± ± ± ± ± ± ±1.67 III 76.02± ± ± ± ± ± ± ± IV 75.09± ± ± ± ± ± V 110.3± ± ± ± ± ± ± ± ±2.86 VI 105.7± ± ± ± ± ± ±2.45 VII 126.4± ± ± ± ± ± ± ± ±4.39 Average of six values in each case ± SD The growth rate of Vigna unguiculata inoculated with rhizobium, PSB and VAM fungus, after 30, 60 and 120 days of growth was studied and the results are given in tables LCIV to LCVI. The no: of nodules and percentage of mycorrhizal infection were increased in the tetrapartiate inoculations than the single ones. As a result of co-inoculation, the number and weight of the nodule and the percentage of VAM infection were increased and that might have resulted in increased growth. Gradual increase in growth rate in all the groups was observed. In vigna plants

11 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 225 inoculated with Rhizobium, PSB and VAM fungi showed three fold increase over control plants after 120 days of growth. Among the different single inoculations, the VAM fungal inoculation showed significant increase than the other single inoculations. Table LCVII: Nutrient uptake levels of Vigna unguiculata, inoculated with Rhizobium, PSB and VAM fungus, after 30, 60 &120 Days of growth % of N % of P % of K 30 DAP 60 DAP 120 DAP 30 DAP 60 DAP 120 DAP 30 DAP 60 DAP 120 DAP I 2.18± ± ± ± ± ± ± ± ± II 4.64± ± ± ± ± ± ± ± ± III 4.71± ± ± ± ± ± ± ± ± IV 3.76± ± ± ± ± ± ± ± ± V 5.38± ± ± ± ± ± ± ± ± VI 4.86± ± ± ± ± ± ± ± ± VII 5.62± ± ± ± ± ± ± ± ± Average of six values in each case ±SD The nutrients uptake (NPK) levels of Vigna unguiculata inoculated with Rhizobium, PSB and VAM fungus, after 30, 60 and 120 days of growth were studied and the results are shown in table LCVII. The VAM

12 226 Chapter 5 inoculated plants showed significant increase in NPK levels than any other single inoculums, except nitrogen. With other single inoculums, VAM showed increased NPK levels. The highest level was observed in tetrapartite association. The rhizobium inoculated plants showed more nitrogen than the other single inoculums. Table LCVIII: Growth rate of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungus after 30 days of growth Shoot weight Plant Shoot - 1 Root gm Root weight length Length (gm) cm Fresh Dry cm Fresh Dry No. of Nodules Weight of Nodule - 1 gm % of mycorrhizal infection I 15.04± ± ± ± ± ± II 28.03± ± ± ± ± ± ± III 23.51± ± ± ± ± ± ± ± IV 24.43± ± ± ± ± ± V 38.81± ± ± ± ± ± ± ± ± VI 36.40± ± ± ± ± ± ± VII 43.62± ± ± ± ± ± ± ± ± Average of six values in each case ± SD

13 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 227 Table LCIX: Growth rate of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungus after 60 days of growth Shoot length Shoot weight gm cm fresh dry Root Length Root weight (gm) cm fresh dry No. of Nodules Weight of Nodule -1 gm % of mycorrhizal infection I 22.03± ± ± ± ± ± II 54.01± ± ± ± ± ± ±2.411 III 45.16± ± ± ± ± ± ± ± IV 40.33± ± ± ± ± ± V 65.41± ± ± ± ± ± ± ± ±2.820 VI 63.83± ± ± ± ± ± ±2.571 VII 77.2± ± ± ± ± ± ± ± ±3.875 Average of six values in each case ± SD

14 228 Chapter 5 Table C: Growth rate of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungus after 120 days of growth Shoot length Shoot weight gm cm fresh dry Root Length Root weight (gm) Cm fresh dry No. of Nodules Weight of Nodule -1 gm % of mycorrhizal infection I 28.16± ± ± ± ± ± II 61.24± ± ± ± ± ± ±3.11 III 50.09± ± ± ± ± ± ± ± IV 51.71± ± ± ± ± ± V 63.42± ± ± ± ± ± ± ± ±4.12 VI 65.21± ± ± ± ± ± ±3.80 VII 76.73± ± ± ± ± ± ± ± ±4.61 Average of six values in each case ± SD Growth rate of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungi after 30, 60 and 120 days of growth were studied and the results are given in tables LCVIII to C. All the groups showed significant increase in growth rate than the control. The co-inoculation of VAM fungus with either Rhizobium or both bacteria showed significant increase and the plants with both Rhizobium and PSB along with VAM fungus showed highest growth rate.

15 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 229 Table CI: Nutrient uptake levels of Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungi, after 30, 60 &120 Days of growth % of N % of P % of K DAP DAP DAP DAP DAP DAP DAP DAP DAP I 1.94± ± ± ± ± ± ± ± ± II 4.19± ± ± ± ± ± ± ± ± III 4.38± ± ± ± ± ± ± ± ± IV 2.92± ± ± ± ± ± ± ± ± V 4.96± ± ± ± ± ± ± ± ± VI 4.86± ± ± ± ± ± ± ± ± VII 5.28± ± ± ± ± ± ± ± ± Average of six values in each case ±SD, DAP- days after planting NPK levels of Arachis hypogeae inoculated with Rhizobium, PSB and VAM fungus, after 30, 60 and 120 days of growth were studied and the results are given in table CI. All the treated plants showed two to three fold increase in nutrient contents except the plants inoculated with PSB, when compared to controls. The phosphorus content in Rhizobium inoculated plants showed the least value among the different treatments. Gradual increase in the level of NPK was observed in all plants compared to controls when they were uprooted after 30 to 120 days of growth. The

16 230 Chapter 5 most significant increase in the NPK level was observed in Arachis hypogeae when it was inoculated with Rhizobium, PSB and VAM fungus. The percentage of infection of VAM fungus was greater in this combination and the no: of nodules were also higher than other treatments Bio chemical constituents Biochemical constituents like total chlorophyll, carbohydrates, reducing sugars and proteins in mycorrhizal and non-mycorrhizal leguminous plants, inoculated with PSB and Rhizobium separately and in mixture were studied in two leguminous plants and the results are given in tables CII to CIX. Table CII: Total chlorophyll in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth Total chlorophyll (mg/gm fresh tissue 30 DAP 60 DAP 120 DAP Significance between groups I 1.270± ± ± II III IV V VI 2.760± ± ± ± ± ± ± ± ± ± ± ± ± ± ± I&II-* I&III-** I&IV-** I&V-** I&VI-** I&VII-** Other interactions-ns VII 3.218± ± ± Average of six values in each case ±SD, * P<0.05, **P<0.01,NS- not significant

17 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 231 The total chlorophyll in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30,60 & 120 days of growth was studied and the results are given in table CII. It was found that among the different inoculations, VAM fungal inoculation was better than the other single inoculations. PSB or Rhizobium inoculations along with VAM showed increased chlorophyll content. Highest chlorophyll content was observed in the tetra partite association of VAM, Rhizobium and PSB in Vigna plants. A progressive increase in the chlorophyll content was observed in all the plants, when they were uprooted after 30 to 120 days of growth when compared to controls. Table CIII: Total Chlorophyll in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth. Total chlorophyll (mg/gm fresh tissue) 30 DAP 60 DAP 120 DAP Significance between groups I 1.760± ± ± II III IV V VI 3.310± ± ± ± ± ± ± ± ± ± ± ± ± ± ± I & II- * I & III- * I & IV- * I & V- ** I & VI- ** I & VII- *** other interactions - NS VII 4.118± ± ± Average of six values in each case± SD,* P<0.05, ** P<0.01, *** P<0.001, NS- not significant

18 232 Chapter 5 Total chlorophyll in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with PSB and rhizobium after 30, 60 and 120 days of growth was studied and the results are given in table CVI. Total chlorophyll content in mycorrhizal plants inoculated with both PSB and rhizobium was found to be increased very significantly, after 120 days of growth than any other treatments. Chlorophyll content was more in VAM infected plants than PSB or rhizobium inoculated plants, but not significant. In Arachis, the dual inoculation did not have significant change in the level of chlorophyll than VAM treatment alone. Increase in chlorophyll was observed in all the groups, from 30 to 120 days of growth. Table CIV: Total Carbohydrates in mycorrhizal and nonmycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth. Total carbohydrate (mg/gm fresh tissue DAP DAP DAP Significance between groups I 3.630± ± ± II III 7.630± ± ± ± ± ± I&II- *** I&III- ** I&IV- ** I&V- *** IV 6.021± ± ± I&VI- *** I&VII- *** V VI 8.693± ± ± ± ± ± II&VII-* III&VIII- ** IV&VII- *** Other interactions NS VII ± ± ± Average of six values in each case± SD, * P<0.05, ** P<0.01, *** P<0.001, NSnot significant

19 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 233 Total carbohydrates in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with PSB & Rhizobium after 30, 60 and 120 days of growth were studied and the results are given in table CIII. Carbohydrate content was increased significantly in mycorrhizal plants inoculated with rhizobium and PSB when compared to other inoculums. VAM fungal inoculum showed high levels of carbohydrates than PSB or Rhizobium inoculations. The PSB and Rhizobium inoculations did not show significant change in carbohydrates content, when compared. Table CV: Total Carbohydrates in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth. Total carbohydrates (mg/gm fresh tissue) 30 DAP 60 DAP 120 DAP Significance between groups I 3.820± ± ± II III IV V VI 7.830± ± ± ± ± ± ± ± ± ± ± ± ± ± ± I & II- *** I &III- ** I & IV- ** I & V- *** I & VI- *** I & VII- *** III & VII- ** IV & VII- ** Other interactions-ns VII ± ± ± Average of six values in each case± SD,* P<0.05, ** P<0.01, *** P<0.001, NS- not significant Total carbohydrates in Arachis hypogeae inoculated with Rhizobium, PSB and VAM fungi, after 30, 60 and 120 days of growth were studied and are given in table CVII. Increased levels of carbohydrates were found in all groups, when compared to control plants. But the

20 234 Chapter 5 Rhizobium or PSB inoculated plants showed no significant difference in carbohydrate contents. The co-inoculation of rhizobium or PSB along with VAM fungus also showed no significant change. VAM plants inoculated with Rhizobium and PSB showed significant increase in the carbohydrates, when compared to any other groups. Table CVI: Total Reducing Sugars in mycorrhizal and nonmycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth. Total reducing sugars (mg/gm fresh tissue 30 DAP 60DAP 120 DAP Significance between groups I 1.280± ± ± I &II- *** II 4.310± ± ± I &III- ** I &IV- ** III 3.924± ± ± I &V- *** I &VI- *** IV 3.615± ± ± I &VII- *** II &VII- *** V 5.244± ± ± III &VII- *** IV &V- * VI 5.019± ± ± IV &VI- * IV &VII- *** VII 6.114± ± ± Other interactions- NS Average of six values in each case± SD, * P<0.05, ** P<0.01, *** P<0.001, NS- not significant The total reducing sugars in mycorrhizal and non-mycorrhizal Vigna unguiculata, inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth were studied and the results are given in table CIV. The mycorrhizal plants showed more reducing sugars than the other bacterial inoculations and controls. The dual inoculated plants also showed increased sugar content than the single inoculums. The mycorrhizal

21 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 235 inoculation along with both PSB and Rhizobium showed significant increase in sugar contents than any other inoculations. Table CVII: Total Reducing Sugars in mycorrhizal and nonmycorrhizal Arachis hypogeae inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth. Total reducing sugars (mg/gm fresh tissue) Significance between groups 30 DAP 60DAP 120 DAP I II III IV V VI VII 1.030± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± I & II- *** I & III- *** I & IV- *** I & V- *** I & VI- *** I & VII- *** II & VII- * III & V- * III & VII- *** IV & V- ** IV & VI- * IV & VII- *** Other interactions-ns Average of six values in each case± SD, * P<0.05, ** P<0.01, *** P<0.001, NSnot significant Total reducing sugars in Arachis hypogeae, inoculated with Rhizobium, PSB and VAM fungi, after 30,60 and 120 days of growth were studied and the results are given in table CVIII. The reducing sugars were significantly high in all groups compared to controls. Plants in-group II showed increased sugar content when compared to group III and IV. Plants in group V showed no change in sugars when compared to group VI, but both showed significant increase when compared to control and single inoculations. Plants in-group VII showed significantly high sugar content when compared to other groups.

22 236 Chapter 5 Table CVIII: Total Proteins in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth. Total proteins (mg/gm fresh tissue) 30 DAP 60 DAP 120 DAP Significance between groups I 4.810± ± ± I &II- *** I &III- ** II III IV 8.320± ± ± ± ± ± ± ± ± I &IV- * I & V- *** I & VI- *** I & VII- *** II & IV- * II &VII- ** III & V- ** V VI VII ± ± ± ± ± ± ± ± ± III &VI- ** III &VII- *** IV & V- *** IV &VI- *** IV &VII- *** Other interactions- NS Average of six values in each case± SD, * P<0.05, ** P<0.01, *** P<0.001, NSnot significant Total proteins in mycorrhizal and non- mycorrhizal Vigna unguiculata inoculated with PSB and rhizobium, after 30, 60 and 120 days of growth were studied and the results are given in table CV. It was found that the proteins increased in all plants, when they were uprooted after 30 to 120 days of growth. Mycorrhizal plants had significant increase in the protein content over the plants inoculated with either PSB or rhizobium, but less than the dual inoculums. The tetra partite inoculations of all the microbes showed very significant increase in proteins than any other treatments.

23 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 237 Table CIX: Total Proteins in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with PSB and Rhizobium, after 30, 60 and 120 days of growth Total proteins (mg/gm fresh tissue) 30 DAP 60 DAP 120 DAP Significance between groups I II III IV V VI VII 5.140± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± I & II- *** I & III- *** I & IV- *** I & V- *** I & VI- *** I & VII- *** II & III- ** II & IV- ** II & VII- *** III & V- *** III & VI- *** III & VII- *** IV & V- *** IV & VI- *** IV & VII- *** VI & VII- * Other interactions-ns Average of six values in each case± SD,* P<0.05, ** P<0.01, *** P<0.001, NS- not significant Total proteins in Arachis hypogeae inoculated with Rhizobium, PSB and VAM fungus, after 30, 60 and 120 days of growth were studied and the results are given in table CIX. Plants with single inoculations showed significant increase in protein content than the control plants. Plants in group V showed no significant change when compared to group VI, but both showed significant increase when compared to other single inoculations. Plants in tetrapartiate associations of VAM, Rhizobium and PSB showed maximum protein content when compared to other groups.

24 238 Chapter Nitrogenase and phosphatases (ACP & ALP) activity The experiment was conducted as mentioned earlier. The two leguminous plants were uprooted after 30,60 and 120 days of their growth. The enzymes nitrogenase, acid and alkaline phosphatases responsible for nitrogen and phosphorus metabolism in plants were estimated as mentioned in chapter: 2 and the results are given below. Table CX: Nitrogenase activity in Mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth. Nitrogenase activity in n moles of ethylene produced hr -1 plant -1 at 30 days 60 days 120 days Average I 214±4 240±6 281±4 245 II 421±7 480±5 520± III 436±4 520±11 552± IV 320±6 380±6 416±6 372 V 520±6 615±4 668±4 601 VI 485±9 580±6 624±7 563 VII 680±6 760±7 818±6 Average Average of 6 values in each case ±S.D, C D values (P=0.05) Between Between Days after planting Between x DAP

25 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 239 Nitrogenase activity in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with Rhizobium and Phosphate solubilizing bacteria, after 30, 60 and 120 days of growth was studied and the results are given in table CX. It was found that the enzyme activity was more in mycorrhizal plants inoculated with both Rhizobium and P.S.B than single or un-inoculated controls. Group VII plants showed three-fold increase over controls. Increased activity was observed in all groups from 30 to 120 days of their growth. Table CXI: Nitrogenase activity in Mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth. Nitrogenase activity in n moles of ethylene produced hr -1 plant -1 at 30 days 60 days 120 days Average I 196±7 220±6 260± II 432± ± ± III 476±6 510±9 525± IV 324±7 432±8 496± V 534±10 596±11 639± VI 502±10 565±11 607± VII 642±13 723±14 806±14 Average Average of 6 values in each case ±S.D, C D values (P=0.05) Between Between Days after planting Between x DAP

26 240 Chapter 5 Nitrogenase activity in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with Rhizobium and Phosphate solubilizing bacteria, after 30, 60 and 120 days of growth was studied and the results are given in table CXI. The P.S.B inoculated plants had less nitrogenase activity than other groups, but showed higher activity than controls. Plants in-group III showed more nitrogenase activity than group II. Group V plants showed increased nitrogenase activity when compared to all groups except VII. Table CXII: Acid Phosphatase activity in Mycorrhizal and non- Mycorrhizal Vigna unguiculata inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth. ACP activity in KA units/100 ml sample at. 30 days 60 days 120 days Average I 1.260± ± ± II 2.180± ± ± III 6.800± ± ± IV 4.620± ± ± V ± ± ± VI ± ± ± VII ± ± ±0.18 Average Average of 6 values in each case ±S.D, C D values (P=0.05) Between Between Days after planting Between x DAP

27 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 241 The level of acid phosphatase (ACP) activity in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with P.S.B and Rhizobium, after 30, 60 and 120 days of growth was studied and the results are given in table CXII. It was found that the enzyme activity was maximum in mycorrhizal plants inoculated with Rhizobium and P.S.B and the least in single culture of Rhizaobium. Even though P.S.B was responsible for phosphate metabolism, more acid phosphatase activity was observed in VAM plants. Dual inoculation of VAM and P.S.B showed two fold increase in the enzyme activity than single inoculated ones. Table CXIII: Acid Phosphatase activity in Mycorrhizal and non- Mycorrhizal Arachis hypogeae inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth. ACP activity in KA units/100 ml sample at. 30 days 60 days 120 days Average I 1.210± ± ± II 5.810± ± ± III 2.890± ± ± IV 3.920± ± ± V 6.340± ± ± VI 6.720± ± ± VII 6.960± ± ±0.19 Average Average of 6 values in each case ±S.D,

28 242 Chapter 5 C D values (P=0.05) Between Between Days after planting Between x DAP Acid phosphatase activity in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with P.S.B and Rhizobium, after 30, 60 and 120 days of growth was studied and the results are given in table CXIII. The enzyme activity was found to be increased maximally in-group VII. Plants in group V and VI also showed increased activity, but not significant when compared. Group II showed an increase in the activity when compared to group III and IV. All the groups showed increased enzyme activity when compared to controls. Table CXIV: Alkaline Phosphatase (ALP) activity in Mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth. ALP activity in KA units/100 ml sample at. 30 days 60 days 120 days Average I 0.830± ± ± II 2.927± ± ± III 1.420± ± ± IV 2.890± ± ± V 4.210± ± ± VI 6.280± ± ± VII 7.260± ± ±0.20 Average Average of 6 values in each case ±S.D,

29 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 243 C D values (P=0.05) Between Between Days after planting Between x DAP Alkaline phosphatase activity in mycorrhizal and non-mycorrhizal Vigna unguiculata inoculated with Rhizobium and PSB, after 30, 60 and 120 days of growth was studied and the results are given in table CXIV. Plants in-group VII showed maximum enzyme activity when compared to other groups. Plants in-group II showed increased enzyme activity than group III and IV. No significant increase was observed in-group V when compared to group VI, but both showed significant increase when compared to single inoculations or controls. Table CXV: Alkaline Phosphatase (ALP) activity in Mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with Rhizobium and Phosphate Solubilizing Bacteria, after 30, 60,120 days of growth. ALP activity in KA units/100 ml sample at. 30 days 60 days 120 days Average I 0.780± ± ± II 2.860± ± ± III 1.080± ± ± IV 1.290± ± ± V 3.280± ± ± VI 3.410± ± ± VII 3.780± ± ±0.11 Average Average of 6 values in each case ±S.D,

30 244 Chapter 5 C D values (P=0.05) Between Between Days after planting Between x DAP Alkaline phosphatase activity in mycorrhizal and non-mycorrhizal Arachis hypogeae inoculated with P.S.B and Rhizobium, after 30, 60 and 120 days of growth was studied and the results are given in table CXV. Dual and tripartite inoculations in these plants showed more enzyme activities than single inoculums. Among the different groups studied, the group VII showed maximum enzyme activity after 120 days of growth. Group VI also showed increased enzyme activity when compared to other groups except VII. Plants in-group II showed significant increase in enzyme activities when compared to other single inoculations. 5.4 Discussion Results indicated that the percentage of VAM infection was increased in tetrapartiate association. The percentage of VAM infection in Vigna unguiculata and Arachis hypogeae in different groups after 30, 60 and 120 days of growth were graphically shown in figure XVIII and XIX. Both leguminous plants showed same kind of VAM infection. Among the bacterial inoculums, Rhizobium enhanced the VAM infection than PSB when inoculated dually.

31 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 245 Figure XVIII: Percentage of VAM infection in mycorrhizal and nonmycorrhizal Vigna unguiculata inoculated with Rhizobium and PSB, after 30, 60 and 120 days of growth Percentage of VAM Infection I II III IV V VI VII Figure: XIX. Percentage of VAM infection in mycorrhizal and nonmycorrhizal Arachis hypogeae inoculated with Rhizobium and PSB, after 30, 60 and 120 days of growth Percentage of VAM Infection I II III IV V VI VII

32 246 Chapter 5 A few reports are available on the effect of tetrapartiate association of VAM, PSB and Rhizobium in leguminous plants. Numerous reports available on dual or tripartite associations of VAM, Rhizobium or PSB in leguminous plants. The results obtained in this study revealed that the single inoculation of mycorrhizal fungus is better than the other single inoculations in leguminous plants. The growth rate and nutrient uptake were more in mycorrhizal plants. Any combination containing VAM fungus showed significant increase in growth and nutrient level. The biochemical constituents like total chlorophyll, carbohydrates, reducing sugars and proteins were increased significantly in tetrapartiate associations in leguminous plants. Green house studies conducted by Cabello et al, (2005) reported that the dual inoculation of a VAM fungus Glomus mosseae and a phosphorus solubilizing microorganism Penicillium thomii in Mentha piperita with or with out rock phosphate, showed a positive effect of these microbes on host plant s growth when compared to control. Rodruez et al, (2005) reported that the combined application of mycorrhizal fungus and rhizobacteria in banana plants, showed significant increase in total fresh weight, aerial dry weight, shoot length, leaf area and leaf mineral contents N P& K than non-treated control bananas. Wu et al, (2005) studied the effects of biofertilizer containing N-fixer, P & K solubilizers and AM fungi on Maize growth, in green house trials reported that the application of the combined inoculums showed significant increase in plant height and biomass and improved assimilation of nutrients by improving soil properties, organic matter content and total N in soil. Artusson et al, (2006) reported that Arbuscular mycorrhizal (AM) fungi and rhizobacteria could interact synergically to stimulate plant growth through a range of mechanisms that include improved nutrient acquisition (Nitrogen and phosphorus bioavailability) and inhibition of fungal plant pathogens. Weber et al (2005) reported that the dual inoculation with Glomus intraradices and Bradyrhizobium in Acacia mangium under aeroponic culture, showed increased growth rate than single or non-inoculated ones.

33 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 247 The higher P concentrations reduced the AM frequencies while lower concentrations of P stimulated the development of AM with out affecting plant development. Schenck and Hinson, (1973) reported that the mycorrhizal infection improved N content in the shoots and seeds of nodulated plants than the non-nodulating ones. Thus nodule function is enhanced by mycorrhizal infection. The plants require relatively large amount of phosphorus for optimum growth, nodulation and nitrogen fixation (Carling et al, 1978). Co-inoculation of Rhizobium sps and VAM fungi had a positive effect on growth and nutrient uptake in nodulating plants. Dual inoculation of leguminous plants with Rhizobium and VAM was found to enhance chlorophyll content and photosynthetic rates in Cyamopsis sp (Neeraj and Ajit Verma, 1995). Rice bean (Vigna umbellate) inoculated with G. fasciculatum and Rhizobium sp in a p deficient soil significantly increased VAM colonization, nodulation and yield of plants (Kaur and Singh, 1985). Joshi et al, (1991) reported that a significantly positive correlation exists between percent VAM infection and nodule number in 60 days old groundnut. Gaur (1990), Kucey (1988) reported that the inoculation of plants with phosphate solubilizing fungi was encouraging the proliferation of other phosphate solubilizing fungi. P and N are the two major plant nutrients and co- inoculation of Nitrogen fixers and Phosphate solubilizers may benefit the plant better than either group of organisms alone (Saxena and Tilak, 1994). Manjunath et al, (1981) reported that mixed inoculation of bacteria and fungi increased the yield of onion to a greater extent than the individual microbial content. Smith and Draft, (1977) reported that mycorrhiza- induced, increase in nitrogen fixing rates in Medicago sativa preceded any effect on plant growth. This suggested the idea that nodules demand phosphate. When phosphate was added, the non-mycorrhizal plants showed higher value of % N as that of mycorrhizal ones.

34 248 Chapter 5 Results in the enzymes assays revealed that the enzymes nitrogenase, alkaline phosphatases and acid phosphatases were found to be increased in tetrapartiate associations. When plants were inoculated with single inoculums, VAM inoculation showed maximum activity of these enzymes when compared to others. In dual inoculums also VAM association showed maximum enzyme activity. This ability suggests VAM as a prominent recommendable microbe for soil fertility. Higher amount of P uptake may also be the result of higher activity of acid phosphatase as the total and specific activity of this enzyme in root extracts from the inoculated seedlings was found to be significantly higher than the uninoculated control (Choudhury et al, 2002) Nitrogenase activity in Pueraria sps increased when the growth phosphate response curve become asymptotic (Waidyanatha et al, 1979). It supports the suggestion that nodule function may be preferentially stimulated by mycorrhizal infection, which makes phosphate directly available to the nodules (Bagyaraj et al, 1979, Dixon, et al, 1993, Mehrotra, 1995 and Priyarani et al, 1999). Bagyaraj and Menge, (1978) reported that the dual inoculation of VAM and Nitrogen fixing bacteria enhance the plant growth, as the nitrogen fixing bacteria produce growth regulators and thus enhancing the root production. The increased root growth might have resulted in more root area for the VAM fungi and Nitrogen fixing bacteria to colonize and thus enhancing the growth. Mosse et al, (1976) found that plants did not nodulate unless their P concentrations were at least 0.15%, mycorrhizal infection helped the plants to reach this required level and nodulation then occurred. Carling et al (1978) found that soluble phosphate can replace VAM in increasing nitrogenase and nitrate reductase activities in nodulated soybeans. Mycorrhizas markedly increase P up take, growth and nodulation in clover at low and intermediate rates of applied P, although plant growth depressions may occur at high levels of available Phosphorus (Crush, 1976). Large P additions to the soil are known to decrease mycorrhizal infection in several legumes. Anguilar and Barea, (1978)

35 Effect of Rhizobium and PSB In Mycorrhizal Leguminous Plants 249 reported that a satisfactory nodulation was greatly dependent on the mycorrhizal symbiosis. Rhizobium and Phosphobacteria cultures improved plant growth, nodulation and mycorrhiza formation. The important benefit of VAM association with leguminous plants is the absorption of phosphates from soil. Legumes generally have less extensive root system as compared to other plants and many of them are poor foragers of soil phosphates. Phosphate is an important macronutrient for leguminous plants due to its role in energy transfer during the process of nitrogen fixation. A good supply of phosphorus is essential for effective nodulation, which could be accomplished by VAM association. Nodules are generally known to possess higher concentration of phosphorus than root tissues. The requirement of P is high in legumes (Bagyaraj, 1991) and there fore leguminous plants respond more to mycorrhizal infection than cereals, which indirectly enhances the biological nitrogen fixation through increased P availability especially in soils with low P content (Sieverding, 1983). Legumes can form two types of associations with microorganisms. One with Rhizobium sp involved in the fixation of atmospheric nitrogen and the other with fungi, that form vesicular arbuscular endomycorrhizas which is concerned with the up take of phosphorus and other nutrients (Crush, 1974). Inoculation of plants with VA mycorrhizal fungi can stimulate nodulation and nitrogen fixation by legumes (Mosse, 1981, Timmer and Leyden, 1980, Giahmi, 1976). Carling (1979) reported that the mycorrhizal nodulated soybean plants exhibited higher levels of nitrogenase and nitrate reductase as compared to non-mycorrhizal plants. This increase in the nitrate reductase system might have a role in increasing the symbiotic effectiveness of VAM. A close association of fungal hyphae with the plant root changes the structure and morphology of the root. It is clear now that the root environment is very complex, where soil and rhizosphere microorganisms and mycorrhizal fungi live and interact with each other. Interactions