MATERIALS AND METHODS

Transcription

1 MATERIALS AND METHODS

2 Materials and Methods The studies pertaining VAM inoculum production and its application in Albizia procera (Roxb.) Benth. were carried out. The experimental site and the plantation o f Albizia procera o f different age groups were selected in Tropical Forest Research Institute, Jabalpur. However, VAM collection from A procera was also made from other localities including TFRI. The experimental work was carried out in Forest Pathology Division laboratory and nursery o f Tropical Forest Research Institute, Jabalpur (M.P.). The site of research work is situated between to N latitude to to to E longitude having annual rainfall of nearly 1383 mm. Annual temperature ranges from 43.2 c maximum to 5.4 c minimum. The soil is red and black clay loam. A systematic survey was made in order to determine the occurrence of VAM both qualitatively and quantitatively associated with A. procera. The samples were taken from different localities (namely TFRI Campus Jabalpur, Coal mine Korba, Iron mine Dalli Rajhara, Bauxite mine Amarkantak, Forest department Plantation (Mohan Bhata) Bilaspur with varying age groups of Albizia procera plantations. Root and soil samples were collected upto a depth o f 10 cm from the rhizosphere soil with five replicates. The samples were collected in fresh polyethylene bags and their openings were tide with rubber bands. The samples were analyzed for VAM fungi, other microbes including Rhizobium and Azotobacter, nutrients and infection in roots of planted tree species within 6 to 7 days after collection. The soil samples collected from rhizosphere were air dried and passed through a 2 mm sieve. Spore population of VAM fungi was estimated in the collected samples by wetsieving and decanting techniques of Gerdemann and Nicolson (1963). A representative portion of 100 g soil samples was suspended in water until all soil aggregates w'ere dispersed and an uniform suspension was formed. The suspension was passed though a stack of sieves with 22, 60, 80, 150 and 300 o f mesh numbers. The residue was suspended

3 in water and decanted, the process was repeated for complete extraction o f spores and sporocarps of VAM fungi. The spores retained on each mesh were pooled and spore suspension was made up to 100 ml for further quantification. The spore suspension was used to quantify the population of VAM fungi in terms o f number o f chlamydospores and sporocarps in 100 g soil. The spore suspension was transferred in whatman filter paper and placed in a moist perti-dish and examined under stereo-binocular microscope for recording the numbers of chlamydospores present on the filter paper. The resting spores were mounted in polyvinyl lacto glycerol (PVLG), and the size, colour, details of the wall layers and the nature o f subtending hyphae were recorded. The viable VAM spores were extracted by sucrose centrifugation technique (Daniel and Skipper, 1982). 100 g. soil samples were suspended in 1 liter o f water. The material was sieved by 1 mm to 45 mm sieve size and washed separately. The content from the sieve (45 mm) were allowed to centrifuge for 4 minute) at 4000 rpm. The supernatant was transferred into 45 mm sieve and dead, over mature VAM spores were separated out. Subsequently tubes were filled by saturated sugar solution and made a suspension by stirring with glass rod and centrifuged for 15 second in 4000 rpm. The supernatant was collected in 45 mm sieve size and washed in tap water. Finally transferred into petri-dish for observation under microscope. Root Colonization The development of VAM fungi in term of root colonization was measured by the method recommended by Phillips and Hayman (1970). The roots of each soil samples (five feeder root < 2 mm dia.) were collected and washed carefully. They were cut in bits, immersed in FAA (Formaldehyde 5 ml, Glacial Acetic Acid 1 ml; Ethanol (95%) 90 ml). The root segment o f 1 cm size was taken in test tube and treated in 10% KOH solution and autoclaved for about 10 minutes. Some o f the samples were cleared by putting the root segments in 10% KOH and heated on a water bath for about minutes. Then the KOH was poured out and the roots were washed in tap water for 3 to 5 times, and

4 acidified with 1% Hcl for 10 minutes to neutralize excessive KOH. After that the acid was poured out, roots were stained in 0.05% trypan blue in lactophenol for 10 minutes. Hundred roots bits were examined under compound microscope and the per cent of root colonization was calculated by the following formulae: No. of infected roots by VAM Per cent of root colonization = x 100 Total no. of root bits observed Total No. of single VAM species Relative density (%) = x 100 Total No. of all the species Identification The VAM isolated, obtained as loose cluster of spores or sporocarps and chlamydospores from soil were thoroughly washed and mounted in PVLG for their identification upto generic or species level. The identification of VAM fungi was confirmed by using the manual of Schenck and Perez (1990) and the synoptic key was useful for confirmation o f VAM species. Isolation of rhizobium Plants were carefully uprooted and the root system washed in running water to remove adhering soil particles. They were immersed in 0.1 per cent acidified HgCl2 for 4-5 minutes. Nodules which were surface sterilized with HgCk were washed repeatedly with sterile water and dipped in 70 percent ethyl alcohol followed by more washing with sterile water. The nodule was crushed in a small aliquot of sterile water with the help of a glass rod. The serial dilutions were prepared from the nodule extracts and aliquot of

5 appropriate dilutions is spread on yeast extract mannitol agar (YEMA). The plates were incubated upto 10 days in an incubator at 26 c. Large gummy colonies of bacteria emerge within 4-5 days. The chemical were used for the preparation of media are mentioned in Annexure -1. Isolation of Azotobacter A weighed sample of soil (log ) was suspended in 90 ml of sterilized water and serial dilution o f the suspension was prepared for further dilutions. Jensen's media was prepared and poured into sterilized petriplates. One ml aliquots of appropriate dilutions are evenly spread over cooled and set agar medium in petriplates. The plates are incubated at 28 c + 2 c in an incubator. After 3 days o f incubation, flat, soft, milky and mucoid colonies o f azotobacter developed on agar plates. Inoculum production of VAM Unlike rhizobia that can be isolated in pure culture and mass multiplied in fermentors for production of legume inoculants, VAM fungi are obligate endosymbionts and scarcely grow in pure culture. This obvious limitation and presently root based bulk inocula the only means o f using VAM fungi in forestry hamper large scale production. After a wet sieving procedure individual type o f living spores are identified and selected under a binocular stereomicroscope. About 50 spores of defiend species are surface sterilized by immersing them in 2 % (w/v) chlromine T and 200 ppm streptomycin for about 15 minutes followed by successive washing in sterile water until the sterilent is removed (Hayman, 1982). These spores are used to infect seedlings grown in sterilized soil The root system is checked microscopically for uniform infection by this selected VAM fungus. The root systems of uniformly well infected seedlings, together with the adhering soil are finally chopped and are used as the starter inoculum to scale up the

6 production of inoculum in bulk by infecting fresh seedlings o f selected plants such as, maize and other grasses raised in sterile soil inoculated with 5-10 % of starter inoculum as a layer approximately 2 inches below the soil level The potted plant kept in green house. The root -based inocula are stored in polyethylene bags. The role of substrates and hosts on VAM inoculum production for Albizia procera The different combinations of substrates viz. soil; sand and soilrite were used in this study. The substrates were autoclaved for 2 hours at 10 psi to avoid contamination in the substrates. The process was continued for 2 times to eliminate soil-borne bacteria and fungi to the maximum extent possible. The sterilized substrates were then filled into plastic bags of 2 kg capacity for experimentation. Seed of Albizia procera were surface sterilized with 4 per cent sodium hypochlorite solution and allowed to germinate on a sterilized filter paper placed in a moist perti-dish and then days old seedlings were transferred in sterilized potting mixture. The spores of each VAM species {Glomus mosseae, G. intraradices and Acaulospora species) were separated into individual spore types and than mixed them. In order to eliminate contamination, the spores placed in perti-dish were surface - sterilized by immersing in 200 ppm. streptomycin solution for 15 minutes (Mosse and Philips, 1971), followed by washing three times in sterile water. The surface sterilized spores of VAM were used as inoculum by placing them in young actively growing lateral roots to encourage growth of germ tube (Mosse, 1962; Hepper, 1981). After 90 days plant growth, number of nodules, spore population and root infection were examined. The spore population was counted by removing a portion of soil for separating the spore by wet-sieving and decanting technique. The effective substrate tested earlier was used for maintaining the cultures of VAM by using Albizia procera as the trap plant. The development of VAM examined after 180 days of inoculation. The culture o f VAM was maintained on trap plant (maize) for conducting different experiments.

7 The different combinations o f sterilized sand, soil and soilrite were used for this experiment. Plastic pots (25 x 9.5 cm size) were filled with substrate. The dominant strains of VAM (Glomus mosseae, G. intraradices and Acaulospora spp.) were selected. The studies pertaining to host plant selection namely; Panicum maximum, Zea mays, Tagetes patula (Marigold) and Sorghum sp. (MP. Chari) were used by growing through seeds. The 20 g (having 200 infective propagules) VAM inoculum of test species inoculated after the emergence of host plant raised through seeds. After 3 months number of spores and VAM infection in root were recorded in each trap plants. Enumeration of infective propagules, optimization of doses and storage of inoculum A study was made for the testing of infectivity o f inoculum by storing at room temperature. The methodology' recommended by Sieverding (1991) was adopted for counting the infective propagules. The following materials were required for this experiments: - Slightly air-dried and sieved (0.5 cm mesh opening) sub sample of inoculum (called SUBSTRATE A). Four kilograms sub sample from the field soil, autoclaved, sieved and completely dried (SUBSTRATE B). Eleven kilograms field soil sterilized in an autoclave, un-sieved material (This is SUBSTRATE C). 50 plastic pots of 250 ml volume. Equipment and materials to stain VAM fungal structures in roots and a stereomicroscope. Arranged the plastic pots in 10 dilution levels and each levels were taken in five replicates. The bases of pots were perforated for the irrigation water to drain away. Each pot was filled with 150 g SUBSTRATE C. Took 100 g o f SUBSTRATE A and

8 determined % moisture by the usual drying techniques. Took 350 g substrate A, and 50 g o f this substrate was poured in each pots which was marked as the first dilution level. Subsequently 85 g o f SUBSTRATE A was mixed with g SUBSTRATE B thoroughly in a plastic bag to prepare the next dilution level. Fifty g o f this mixture (85 g. SUBSTRATE A g SUBSTRATE B) was applied in each 5 cups and was marked second dilution level (4'1). Again took the 85 g material from the rest of previous dilution level (4 1) and mixed with 255 g SUBSTRATE B and its 50 g was applied separately in each 5 cups and was marked as third dilution level.. 50 g substrate each pots of dilution level. The process was continued till last dilution level (i.e.4 '9) Thoroughly clean of plastic bags were used for the preparation of each dilution. Finally each dilution level in all 50 cups were covered separately with 50 g of SUBSTRATE C. The five seeds of Zea mays were sown in each cup. After 8 weeks harvested the plants o f all dilutions. The entire soil volume along with the roots was removed from the cups, while the shoot was cut 1 cm above the soil level. The top soil and basal portion of the soil mass obtained the pot was removed. The root in the middle part o f the soil mass were taken out and cut into small pices. The roots were carefully washed and stained with trypan blue. The stained roots were observed under the stereomicroscope and points o f infection were marked as (+) whereas, uninfected roots were marked as (-) sign. The number o f infective propagules was calculated using this formula: - Log 0 = x. Log a - K where 0 is the number o f infective propagules x is the mean number of cups with infection Total number of infected cups Hence x = Number o f replicates per dilution Y = S - X, Where Y - is required to define the K value from the Fisher and Yates table, S - is the number of dilution levels (where the 4 level is the first level), a - is the factor of dilution which is 4 in the case of the fourfold dilution, K - is found in Table VIII Fisher and Yates (1970) at the determined values o f x or y.

9 Polythene bags of 25 x 9.5 cm size were filled with soil + sand + soilrite (1:1:2 by volume). In order to optimize in doses of inoculum, following treatments in five replicates were given: - T, Substrate + 25 g Glomus mosseae T2 Substrate + 25 g G. intraradices t 3 Substrate + 25 g Mixed VAM fungi (Glomus mosseae, G. intraradices, Acaulospora spp. and Gigaspora spp.) t 4 t 5 T6 T7 Tg T9 T 10 Substrate + 50 g Glomus mosseae Substrate + 50 g G. intraradices Substrate + 50 g Mixed VAM fungi Substrate + 75 g Glomus mosseae Substrate + 75 g G. intraradices Substrate + 75 g Mixed VAM fungi Control (Substrate only) One month old seedlings of Albizia procera previously grown in sterilized substrate were transplanted in polypots. The 25 g inoculum contained 255 Chlamydospores of VAM, while mixed VAM contained approximately 280 Chlamydospores. After 6 months of VAM inoculation, plants were harvested and growth parameters like shoot height, root height, fresh weight, number o f nodules, root infection, number o f spores /1 0 0 g soil were estimated. For the storage of VAM culture above ground part o f plant is cut and the soil is allowed to dry. The soil with root is homogenized by chopping with a knife. Then the material is transferred in 500 ml culture bottles. The plastic bottles were stored at room temperature and freeze. For the testing of viability o f inoculum an experiment was conducted using maize as a trap plant. For this experiment two liter plastic pot were filled with sterilized soil+sand ( 1:1) up to % and applied 2 0 g o f inoculum (containing about 600 infective propagules) which stored at room temperature and freeze from December 1998 to April The inoculum storage 1-4 months was inoculated separately in the pots in

10 which sterilized maize seeds were sown. The experiment was maintained up to 8 weeks and number o f spores, per cent colonization by VAM fungi were recorded in table 8. Suitability of VAM inoculum for A. procera. Selection of suitable inocula o f VAM was made in pot experiment. The capacity o f plastic pots, which were used for this experiment were of 5 liters and filled with mixture of sand+soil (1:1 v/v). The seeds of A. procera were sown after proper treatments. The experiment was setup in randomized design. Each treatment was made in triplicate. The experiment was conducted for 4 months. In one treatment 250 spores were applied. Before the application of spores they were surface sterilized by immersing in 200 ppm streptomycin solution for 15 minutes (Mosse and Philips, 1971). Second treatment was made with 100 root bits of la cm in each pot while third treatment was given by taking 25 g of soil inocula having spores and root bits, etc. Control was also setup simultaneously. After 4 months the shoot length, root length, per cent of root colonization and number of spores were observed. Effect of different mixture of compost on VAM infection in Albizia procera An experiment was conducted to study the status o f VAM in the root trainers by using Albizia procera as the test species. The combinations o f treatments are as follows: - 1. Pure compost 2. Compost + Sand (80: 20) 3. Compost + Sand (50: 50) 4. Sand The experiment was conducted in 300 cc root trainers. The compost contained a very less/negligible quantity of VAM spores. The seed of Albizia procera were sown in root trainer after proper treatments. The VAM culture was applied after initiation o f seed

11 germination (after 10 days). VAM culture containing root bits and soil was used. The inoculation was made by using the mixed VAM culture prepared from Albizia procera by using Panicum maximum as the trap plant. The inocula was 25 g/root trainers with a capacity o f 300 c.c. having 250 infective propagules. The culture of Rhizobium (5 ml, 106) was applied in each pot. Infective propagules include the species of Gigaspora, Scutellospora, Glomus mosseae, G. intraradices and Acaulospora scrobiculata. The ph o f the potting media was measured. The experiment was conducted for three months. The VAM colonization in roots of Albizia procera was obtained by using the techniques of Phillips and Hayman (1970). Shoot height, number of nodules and No. of spores/100 g were also measured. Effect of different moisture regime alongwith VAM fungi and fertilizers on growth ofalbizia procera An experiment was conducted in poly bags with a diameter of 9.5 cm and a height 25 cm. The polybags were filled with the soil mix in the ratio of 1:3 (Sand + soil by volume). The experiment was conducted in TFRI nursery. Seeds of Albizia procera were collected from a single tree in TFRI Campus, weighed, selected for uniformity and treated with overnight soaking. The seeds were sown in polyethylene bags. After germination single seedling was maintained in each pot. Twenty five gram o f VAM inoculum was placed 3 cm below seed as pad. Ten ml. broth culture of rhizobium (isolated from the nodules o f Albizia procera) containing 109 rhizobium bacteria per ml was given to each seedlings as soil drench. The fertilizers N (@ 25 kg/ha as urea) and P (@ 50 kg/ha as single super phosphate) were also applied to individual seedling after seed sowing. The seedlings were kept on a clean polyethylene sheet at nursery site. After seed sowing the seedlings of all the treatments received regular watering upto one month. Thereafter, the watering was regulated to maintain 5 different moisture levels. Seedlings of each treatment were divided in five categories. The first category received 100 ml water

12 daily, the second on alternate day and the third, the fourth and the fifth categories received 100 ml water after 2,3, and 4 days interval respectively. In this way rhizosphere soils of 1-5th categories seedlings maintained moisture levels 20%, 16%, 12%, 8% and 4% respectively. This water schedule was followed for 2 months. After 3 months of sowing (2 months under different moisture levels) the seedlings were uprooted by removing the poly bags carefully. The roots were gently washed with water to remove the soil. The total fresh weight, shoot length, root length, per cent of survival and VAM infection per cent were recorded by slide method (Giovannetti and Mosse, 1980). Effect of different P level on VAM development in Albizia procera. The polyethylene bags (25 x 9.5 cm) were filled with the soil mix in the ratio of 1:2:1 (Sand + Soil + soilrite). The weight of soil mixed in a bag was about 2 kg. The seeds of Albizia procera were grown after suitable treatment and experiment was conducted in nursery condition with 3 replication (5 plants in each replicate). The experiment was setup in the randomized block design. After germination only one plant was maintained in each bag. The inoculum of VAM measuring 25 c.c. having 250 infective propagule was used. On the following day single super phosphate at different levels viz. 200 mg, 250 mg, 300 mg and 360 mg was applied in each bag. The inoculation o f VAM was given after the germination of the seedling during the time of the singling o f the seedling in the poly bags. The observations were taken after 120 days after inoculation of VAM and application of phosphorus. The following parameters were considered for observation in this experiment. 1. Shoot length (cm) 2. Root length (cm) 3. Fresh weight of shoot (g) 4. Fresh weight of root (g) 5. Root infection (%) 6. No. o f spores/100 g soil

13 Effect of VAM Hlongwitu bactci ia! and inorganic fertilisers on Albizitt procera. The experiment was conducted in poly pots 25 x 9.5 cm sizes. The poly pots were filled with the soil mix in the ratio 30:30:40 (Sand, soil and compost). The experiment was conducted in TFRI nursery. The seeds for the experiment were collected from a single elite (Phenolypically superior) tree of Albizia procera planted in paddy fields near Bilaspur (M.P.). The collected seeds were graded by shorting out the mal formed and infected seeds. Before sowing the seeds, they were soaked in ordinary sterilized water for 24 hours. Two seeds were sown in each plastic bag during the month of July After germination only one plant was maintained. The nursery experiment was setup in the randomized design with three replicate (25 plants in each replicate) for each of the fourteen treatments. Thus each treatment had 75 plants. To obtain the inoculum of indigenous VAM fungi from the rhizosphere of the previously planted Albizia procera trees (mostly dominated Glomus mosseae, G. intraradices, Acaulospora spp., Gigaspora and Scutellospora spp.) were used as an inocula. The isolated VAM were cultured and maintained on maize plants (Zea mays). The strain of rhizobium was isolated from Albizia procera by adopting the standard procedure described by Vincent (1970). The culture were routinely maintained on YMA (Yeast extract manitol) agar medium. The quality of the rhizobium was maintained by adopting the standard procedure. The cultures of Azotobacter were also isolated from the soil where the Albizia procera trees were growing. The cultures were maintained in the synthetic medium. Its liquid culture having the bacterial population 106 was taken as the rhizobial inoculant. Ten ml of the culture was drenched in each poly bags. The inoculation of VAM (25 g), rhizobium and Azotobacter (10 ml) was given after the germination of the seedlings particularly at the tune of singling the seedling in poly bags. The nitrogen and phosphorus singly and in combination were given to the plants in 3 different doses i.e. 1 N

14 (180 mg), 2 N (360 mg), 3 N (540 mg) and 1 P (250 mg), 2 P (500 mg), 3 P (750 mg). The treatments o f inorganic fertilizers were given at the same time when bio-fertilizers were applied. All measurements were taken after the six months o f treatment. The growth parameters including collar diameter, height growth of the seedlings, fresh and dry weight of the plants and nodule numbers were recorded. All observations were statistically analyzed. Effect of VAM, Rhizobium and inorganic fertilizers on growth of Albizia procera in microplot and in field For this experiment micro-plots were used 30 x 30 cm sizes. After the preparation of microplots seeds o f Albizia procera were sown after soaking 24 hours in ordinary water. The nursery experiment was setup in randomized block design with three replications (25 plants in each replicate) for each of the 11 treatments. Thus each treatment has 75 plants. All treatments were given immediately after the germination o f seeds in each plot. The treatments with single super 20 kg ha'1, Nitrogen 75 kg ha'1 (as urea), rhizobium 10 ml having 106 bacterial population, mixed VAM inoculum 25 g having 250 infective propagules were applied. In all eleven treatments were given with bio fertilizers or inorganic fertilizers. Seedlings were harvested after 3 months of inoculation and growth, biomass, number o f spores, infection percent o f VAM and number of nodules were estimated. A field experiment was conducted by inoculation o f mixed VAM fungi, Rhizobium, Azotobacter, as well as inorganic fertilizers and their different combinations were also applied. Albizia procera was used for this study, which were planted in the

15 month o f September 1989 on TFRI campus. Pits size 30 x 30 x 30 cm was executed at lm x lm spacing. No soil amendment was done in pits and 4 months old seedlings were used for plantation. The experiment was arranged in randomized block design. The each treatment was given with replication having eight plants in each replicate. Each plant was cut on ground level after establishments during December 1998 in order to maintain uniform height. Inoculation of VAM was made by putting VAM in the rhizosphere o f seedlings planted in TFRI nursery. The mixed VAM containing Glomus mosseae, G. intraradices, Acaulospora spp., Gigaspora spp. were the rate of 30 g inoculum having 300 infective propagules. 10 ml as broth culture having bacterial population 106 was tried. Azotobacter was 10 ml having 106 bacterial population. While N was 75 g per pit with 2 split doses and single super phosphate 25 g per pit. The observations were made after one year of the treatments. The plants were harvested and height, Collar diameter and fresh weight were recorded. The VAM infection percent and the spore population were also estimated from roots and soils o f inoculated plants. Soil Chemical Analysis Organic matter and carbon were estimated by method described by Black (1965), while available nitrogen was estimated by rapid titration method (Subbiah and Asija, 1956). The ph and electrical conductivity were measured in 1:2:5 soil water ratio with glass electrode, ph and conductivity meter. Statistical Analysis of Data The experimental plants in case of pot and field experiment were arranged in randomized block desiga The data in all cases was subject to analysis of critical differences and standard error. This all was made by using computer in Sx programme.