Phyllosphere of Cotton as a Habitat for Diazotrophic Microorganisms

M. G. Murty
1984 Applied and Environmental Microbiology  
Positive nitrogenase activities ranging from 0.18 to 0.78 nmol of C2H4 cm-2 h'were detected on the leaf surfaces of different varieties of cotton (Gossypium hirsutum L. and G. herbaceum L.) plants. Beijerinckia sp. was observed to be the predominant nitrogen-fixing microorganism in the phyllosphere of these varieties. A higher level of phyllosphere nitrogen-fixing activity was recorded in the variety Varalaxmi despite a low C/N ratio in the leaf leachates. Leaf surfaces of the above variety
more » ... e above variety possessed the largest number of hairy outgrowths (trichomes) which entrapped a majority of microbes. Immersion of plant roots in nutrient medium containing 32p, led to the accumulation of label in the trichome-borne microorganisms, thereby indicating a possible transfer of nutrients from leaf to microbes via trichomes. Extrapolation of acetylene reduction values suggested that 1.6 to 3.2 kg of N ha-' might be contributed by diazotrophs in the phyllosphere of the variety Varalaxmi during the entire growth period. Colonization of leaf surfaces of plants by a diverse array of microorganisms is now well documented (12, 18). A considerable number of nitrogen-fixing organisms have been reported to occur in the phyllospheres of a wide range of plants (19, 24, 28) . These organisms, utilizing carbohydrates excreted by the leaves, fix appreciable amounts of nitrogen which might benefit the plants (19) . Investigations in recent years have confirmed the above-mentioned observations in the phyllospheres of mulberry (28), Douglas fir (9), maize and Guatemala grass (3), and rice (8). High fixation rates have been reported also on the phyllosphere of Spartina alterniflora (7) and some other C3 and C4 graminaceous plants (13). The information available on phyllosphere nitrogen fixation and its actual contribution to economically important crop plants is scanty and fragmentary, particularly with respect to the mode of nutrient exchange between the plant and the microorganism. In this report, a comparison of acetylene reduction (AR) activities on the leaf surfaces of different varieties of cotton (Gossypium hirsutum L. and G. herbaceum L.) plants and the contribution made by nitrogen fixers in one of the varieties, namely, Varalaxmi, are made. (Table 1) were obtained from pot-grown plants maintained in the open yard (temperature, 30 ± 2°C) of a nearby nursery. MATERIALS AND METHODS Plant material. Leaves of different varieties of cotton Each pot contained 6 kg of a 2:1:1 mixture of soil-sandfarmyard manure for two plants. The pots were watered regularly to 60 to 80% water-holding capacity. Unless otherwise stated, all plants were in the preflowering to flowering stage at the time of experimentation. The samples were collected in the late afternoon to allow maximum accumulation of photosynthate and were quickly brought to the laboratory in sterile petri dishes placed in ice. Enumeration of nitrogen-fixing bacteria. The nitrogenfixing bacteria from the leaf surfaces were estimated either by impressing the leaves for 5 min on Burk's modified nitrogen-free solid agar (27) as reported earlier (4) or by plating the serially diluted leaf washings (28) on the abovementioned medium. The microflora trapped on the membrane filters (Millipore Corp.) were determined by serial 713 dilution and plating of the membrane washings as stated above. Values were expressed on a dry-weight basis (oven dried at 80°C for 24 h) or on a leaf area basis (measured by planimeter). AR measurements. Rates of nitrogen fixation on surfaces of detached leaves were estimated in 50-ml Erlenmeyer flasks sealed with rubber bungs fitted with serum stoppers as described earlier (13) , using the AR technique (16). The in situ experiments were conducted with pot-grown plants by enclosing the shoots in 1-liter polyethylene bags (Fig. 1) . The bags were checked for leaks by pressing the aerated bags under water. Freshly generated C2H2 was then injected through the air-tight bag to make a 10% (vol/vol) acetylene atmosphere inside the bag. The incubations were carried out for 2 h under field conditions. Neither evacuation nor flushing of the bags with argon was done. The bags were kneaded gently so that the gases inside could be mixed well. Needle punctures made during injection were sealed immediately with a quick-drying sealant. At the end of the incubation period, the bagged pots were carefully returned to the laboratory and the C2H4 produced was measured by gas chromatography. Acetylene blanks and plants minus acetylene were always included for determination of background ethylene. Ethylene was measured in a Perkin-Elmer model Fli gas chromatograph fitted with a hydrogen flame ionization detector and a Porapak N column (125 by 0.15 cm). Nitrogen gas (1 kg cm-2) served as carrier gas. The oven temperature was adjusted to 70°C. The values were normalized for dry weight as well as for leaf area. In situ collection of leaf leachates and C/N estimation. Plastic troughs of 20-cm diameter were carefully fixed to the bases of plants through narrow slits, and the leachates were collected into the troughs by applying a fine spray of distilled water (200 ml each time) to the leaves with an atomizer sprayer. Leachates were collected and pooled from two test plants at three points in the diurnal cycle, i.e., at 9, 12, and 15 h. The volume of leachates in each case was separately reduced to 5 ml by lyophilization and membrane filtered (0.45-,um pore size; millipore) to eliminate any microorganisms. The carbohydrates in the leachates were determined colorimetrically by the anthrone method (29), and ninhydrin-on May 8, 2020 by guest http://aem.asm.org/ Downloaded from
doi:10.1128/aem.48.4.713-718.1984 fatcat:lurpvdkjjre47nvvofp6erudqm