Published in Agron J 99:1260-1264 (2007)
DOI: 10.2134/agronj2006.0367
© 2007 American Society of Agronomy
677 S. Segoe Rd., Madison, WI 53711 USA
Mycorrhizae
Arbuscular Mycorrhizal Fungi Associated with Green Gram in South India
N. Valsalakumara,
J. G. Raya,* and
V. P. Pottyb
a Environment Science Research Lab., St. Berchmans College, Changanacherry, Kerala, India, 686 101
b Central Tuber Crops Research Institute, Sreekaryam, Thiruvananthapuram, Kerala, India
* Corresponding author (methikalamray{at}yahoo.com)
Received for publication December 28, 2006.
 |
ABSTRACT
|
|---|
Field studies are necessary to understand the abundance and type of indigenous arbuscular mycorrhizal (AM) fungi present in the rhizosphere. Green gram [Phaseolus aureus Roxb. (= Vigna radiata var. radiata)] is a major pulse crop of South India. A field study was conducted to identify the AM fungi associated with green gram under field conditions in this region. The rhizosphere soil samples from these fields were analyzed for AM fungal spores. Glomus mosseae, Glomus microcarpum, Gigaspora margarita, and Scutellospora sp. were identified as the AM fungi associated with green gram. Glomus mosseae was the most frequent AM fungal associate identified in 81%, G. microcarpum and G. margarita in 24% each, and Scutellospora sp. in 5% of the soils studied. The range of distribution varied from a single species of AM fungus to three species belonging to two genera in one sample. Correlations of spore count and percentage colonization for all the AM fungi on the crop in the field, along with soil parameters like pH, N, P, and K were studied. Significant negative correlation (P < 0.01) was obtained between percentage colonization and P. The pH and N were positively correlated (P < 0.01) with K. The study indicates involvement of factors other than soil nutrients in the distribution and sporulation of AM fungi.
Abbreviations: AM, arbuscular mycorrhizal
|
INTRODUCTION
|
|---|
GREEN GRAM IS A PULSE CROP, with many nutritional and medicinal properties. In India its cultivation is limited to certain localities. However, it is a promising crop to meet the protein requirements of future generations. To meet the present global demand, the production of this crop has to be improved by adopting sustainable technology. Possibilities for mycorrhizal associations in improving crop production are being investigated in many crops. Arbuscular mycorrhizal fungi have been investigated for their potential as synergists in crop plants (Ikram, 1990; Sullia et al., 1991; Terry et al., 2002). The beneficial effect of indigenous AM fungi on the nutrition of agricultural plants depends on both the abundance and type of fungi present in the soil (Abbott and Robson, 1982). However, the potential for employing AM fungi on a wide scale in agriculture is dependent on the development of crop-growth-promoting strains of AM, which are superior to native soil population of AM fungi (Menge, 1983). A field study is therefore necessary to understand the abundance and type of indigenous AM fungi present in the rhizosphere of the crop. Various factors such as fertilizers, season, soil type, cropping system, and fungicides can influence sporulation in AM fungi (Hayman, 1970; Land and Schonbeck, 1991; Land et al., 1993; Sugavanam et al., 1994) which can be revealed in correlation studies.
Rao and Rao (1996) conducted an experimental study on the effect of AM fungi on the roots of green gram as influenced by different sources of phosphorus. Thakur and Panwar (1997) reported a combined influence of Rhizobium and arbuscular mycorrhizae on photosynthesis, N metabolism, and sucrose translocation in Phaseolus radiatus. Similar effect of AM fungi on green gram was reported by Das et al. (1999) and Hazarika et al. (2000). However, a description of the native AM flora on green gram in South India is not available. With this in view, the present study was undertaken to identify the native AM fungi associated with green gram cultivated in different localities of two South Indian states, Tamilnadu and Karnataka. Three genera of AM fungi were usually found associated with the crop. Glomus mosseae was the most frequent mycorrhizal associate. Correlations of spore count and percentage colonization for all the AM fungi on the crop in the field, along with soil parameters of the cultivated sites in South India, are described.
|
MATERIALS AND METHODS
|
|---|
Studies were conducted in the major pulse growing regions of Tamilnadu and Karnataka. The sampling locations were selected at random with a minimum distance of 10 km in between. The sampling sites S1 to S15 were in four Districts of Tamilnadu (Tirunelveli, Teni, Dindigal, and Coimbatore) and S16 to S21 were in Chamarajnagar District of Karnataka (Fig. 1
).
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1. Location map. S1, Ambalavanapuram; S2, Valliyur; S3, Ambasamudram; S4, Alangulam; S5, Thevaram; S6, Dwarasamipuram I; S7, Dwarasamipuram II; S8, Puthupetty; S9, Periyakulam I; S10, Allinagaram; S11, Periyakulam II; S12, Sukkumanaikampetty; S13, Oddanchathram; S14, Thalayathu; S15, Udumelpetta; S16, Gundalpet I; S17, Gundalpet II; S18, Gundalpet III; S19, Gundalpet IV; S20, Gundalpet V; S21, Gundalpet VI.
|
|
Soil samples from the rhizosphere zone of green gram at a depth of 20–30 cm were collected from 21 sampling sites during October–November 2005. Two samples were collected from each sampling site spaced within 10 m and mixed to form a composite sample. All the samples were collected when the plants were either in blossom or bearing fruit. From each spot in a sampling site, a plant along with a cylinder of soil was dug out without damaging the root system. The feeder roots in the soil sample from two spots at a sampling site were collected together and fixed in 10% potassium hydroxide (KOH) for assessment of percentage colonization. The soil left after collecting the roots was mixed and stored in plastic bags and used for extracting spores as well as for studying soil fertility parameters. All the analyses were completed within 1 wk from the date of sampling.
The soil samples were analyzed for AM fungal spores, following a wet sieving and decanting procedure (Gerdemann and Nicolson, 1963), and the spore counts were performed by plate method described by Smith and Skipper (1979). One gram of moist soil was added to 9 mL of distilled water in a test tube capped with a rubber stopper. The tube was then vigorously agitated by hand and 1 mL was immediately pipetted in parallel lines onto a 9-cm filter paper disc in a Petri dish. The filter paper was then scanned wet under a dissecting microscope and the spores were counted. The spore count was multiplied upward to represent 100 g of soil. Ten plates were prepared from one soil sample and the data were represented as average. Identification of AM fungi was based on the system proposed by Hall and Fish (1979). The spores were identified from the plate for spore count and the number of each species was determined. It was then multiplied upward to represent 100 g of soil. Estimation of mycorrhizal colonization of the root system was done after clearing and staining the roots (Philips and Hayman, 1970). The roots were cut into pieces of 1-cm length and boiled in 10% KOH and washed with water. After washing, the root segments were neutralized with 10% hydrochloric acid (HCl). The clear root segments were stained with 0.05% trypan blue in lactophenol. The root segments were destained after 2 h using lactophenol and examined for AM fungal colonization. Presence of hyphae, vesicles or arbuscules was taken as positive indication for colonization. One hundred root segments per soil sample were examined for assessing mycorrhizal infection. The results were expressed as percentage colonization.
The soil from different sampling sites was analyzed for soil fertility parameters such as pH, N, P, and K. Measurement of pH was done using pH meter (Micro pH system 362 Systronics) in 1:2.5 (soil–water) ratio (Byju, 2001, p. 12–15). Micro diffusion method (Sparks, 1996, p. 1358) was employed for quantifying N released as ammonia. Two grams of powdered soil was taken in an injection bottle and 2 mL of 0.32% KMNO4 and 2 mL of 40% NaOH solutions were added. After mixing well, the bottle was kept inside a plastic bottle, which contained 10 mL boric acid-mixed indicator. The plastic bottles were tightly closed and kept in an incubator for 18 h at 38°C. The injection bottles were removed after the release of N as ammonia from the soil. The bottles were rinsed from outside with distilled water into the same plastic bottle. The ammonia absorbed by boric acid resulted in bluish color. It was titrated against 0.005 M H2SO4.
The phosphorus was estimated by the Bray and Kurtz (1945) method using a spectrophotometer (Visible spectrophotometer-Visican 167 Systronics). Flame photometry (Flame photometer Model 128 Systronics) was used for estimation of potassium by leaching the soil in neutral 1N ammonium acetate solution (Jackson, 1962). The correlation studies were conducted using Minitab software.
|
RESULTS AND DISCUSSION
|
|---|
Identification of Arbuscular Mycorrhizal Fungi
The AM fungal spores in the rhizosphere of green gram were identified as belonging to three genera: Glomus, Gigaspora, and Scutellospora. Two species of Glomus (G. mosseae and G. microcarpum), and one species each of Gigaspora (G. margarita) and Scutellospora (Scutellospora sp.) were identified. Glomus mosseae was the most frequent mycorrhizal associate, while Scutellospora was found only in one sample (Sample 20). In Samples 8, 9, 11, 14, and 17, spores of G. mosseae and Gigaspora margarita were identified, although the spore count of the latter was very low. Glomus microcarpum was identified in Samples 2, 4, 15, and 20, along with Scutellospora. In Sample 11, G. microcarpum was identified along with G. mosseae and Gigaspora (Table 1). Native AM fungi in green gram showed low species diversity in the present study. Few spores were noticed in the sieving, whose identity could not be traced from morphological features. However, this was the first study of AM fungi associated with green gram in field soils of South India, where the plant has been a regular crop for many decades.
The present study was based on a single composite sample from each plot. Variability within one location could not be traced in this way. But almost uniform AM fungal diversity was observed in distantly located sites. This suggests that the spatial factor had only narrow influence on AM fungal diversity in green gram.
Percentage Colonization
Percentage colonization of AM fungi in root samples showed lowest colonization (28%) in Sample 8 and the highest (95%) in Sample 4. Out of 21 samples, 15 showed colonization above 70% (Table 1). The degree of colonization was found to vary from 18–98% in various pulses, including green gram in eastern Uttar Pradesh in North India (Hasan, 2002). A negative correlation (–0.624) between P content and percentage colonization was observed in this study, agreeing with previous findings in other crops (Amijee et al., 1989; Douds and Schenck, 1990).
Spore Counts
The total spore count in 100 g soil ranged from 760 to 3920. The lowest spore count was recorded in Samples 16 and 17, while the highest count was in Sample 11 (Fig. 2
).
The highest spore count for individual AM fungus was recorded for G. microcarpum (3560 spores 100 g–1 soil) in Sample 15 and the lowest for G. margarita in sample 17 (10 spores 100 g–1 soil). Apart from soil fertility parameters, nature of the previous crop might have a major influence on AM fungal diversity and sporulation (Diaza et al., 1992). Since green gram is raised as an interim crop between two major crops in all the sites studied, the influence of previous crops on the AM fungal diversity and percent colonization might be possible. This factor was not assessed in the present study.
Soil Parameters
The range of soil pH observed in the field was from 5.3 to 6.8 (Table 1). Arbuscular mycorrhizal fungi vary in their tolerance to pH, and sporulation in certain species decreases with increase in pH (Green and Graham, 1976; Clark, 1997; Sidhu and Behl, 1997). But no significant correlation was observed between spore count and pH. Nitrogen levels ranged from 95.65 kg ha–1 (Samples 11, 12, and 13) to 353.90 kg ha–1 (Sample 18). As per Muhr et al. (1965), 18 soil samples (86%) showed low N availability (<280kg ha–1) and three soil samples (14%) showed medium N (280–560 kg ha–1) content. No significant change in percentage colonization and spore count for Glomus mosseae, Glomus microcarpum, and Gigaspora margarita were found in soils with low and medium N. Scutellospora was found only in soil with medium N content.
Phosphorus levels ranged from 6.52 kg ha–1 (Sample 3) to 150.84 kg ha–1 (Sample 5). It is well known that medium P availability is critical to AM symbiosis, and that percentage colonization among different species varies widely at a fixed level of P (Bagyaraj et al., 1999). In this study at Site 11, where P content was very low (<10 kg ha–1), the highest species diversity, percentage of colonization, and spore counts were observed. Similar observations were reported by Hayman (1983) and Sylvia and Neal (1990). However, the higher rate of colonization in other soils with medium (10–25 kg ha–1) to high P content (>25 kg ha–1) suggests that AM colonization of root is influenced by factors in addition to P. Arbuscular mycorrhizal fungal diversity was found quite low in fields of green gram in South India, which may be attributed to conventional tillage. Jansa et al. (2002) reported a similar reduction in the diversity of AM fungi and predominance of Glomus sp. in a temperate soil affected by tillage.
The potassium content of soil samples ranged from 120.40 kg ha–1 (Sample 15) to 1413.44 kg ha–1 (Sample 19). The K content of the soils was rated as medium (120–280 kg ha–1) to high (>280 kg ha–1). Although K content of the soil and AM fungal distribution are not usually considered to be closely related, mycorrhizal association enables better uptake of this nutrient (Marschner and Dell, 1994). Glomus mosseae was found to be more related to improved K nutrition than P nutrition in soybean [Glycine max (L.) Merr.] (Bethlenfalvay et al., 1989), and hence the predominance of G. mosseae in the fields of green gram in South India may be beneficial to K nutrition of this crop also.
Distribution of Arbuscular Mycorrhizal Fungi
G. mosseae and G. microcarpum were the most common AM fungal associates with P. aureus in the agricultural soils studied. Four field soil samples showed spores of more than one AM fungus. It is possible that the same root may become infected by more than one AM fungus (Abbott and Robson, 1982).
Arbuscular Mycorrhizal Fungi and Nutrient Range
The nutrient range for the AM fungi associated with green gram is presented in Table 2. The nutrient range of G. mosseae and G. microcarpum was much wider than that of Gigaspora and Scutellospora. The wide range of nutrient tolerance may be one reason for the wide spread in occurrence of the genus Glomus. However, the results of correlation studies indicate that AM fungal colonization and sporulation are not merely controlled by soil nutrients as was suggested earlier by Hayman (1970).
Results of Correlation Studies
The results of correlation studies between soil fertility parameters, spore count, and percentage colonization are summarized in Table 3.
|
CONCLUSIONS
|
|---|
The field study on endomycorrhizal associations in P. aureus in South India revealed the presence of two species of Glomus and one species each of Gigaspora and Scutellospora. The genus Glomus was found to be the frequent AM fungal associate with green gram, either singly or in combination with other AM fungi. Since G. mosseae and G. microcarpum are the frequent AM fungal associates with the crop under study, the effect of individual AM fungal species and the consortium of the two species requires further investigation. These studies are presently being conducted.
|
ACKNOWLEDGMENTS
|
|---|
We acknowledge the UGC (University Grants Commission), New Delhi for the financial assistance for carrying out the work.
|
REFERENCES
|
|---|
- Abbott, L.K., and A.D. Robson. 1982. The role of VAM fungi in Agriculture and the selection of fungi for inoculation. Aust. J. Agric. Res. 33:389–408.[Web of Science]
- Amijee, F., P.B. Tinker, and D.P. Stribley. 1989. Development of mycorrhizal root system. VII. A detailed study of effects of soil P on colonization. New Phytol. 111:435–446.[Web of Science]
- Bagyaraj, D.J., A.N. Balakrishna, and B.J.D. Reddy. 1999. Biodiversity in arbuscular mycorrhizal fungi. p. 359–367. In D.J. Bagyaraj et al. (ed.) Modern approaches and innovations in soil management. Rastogi Publ., Meerut, India.
- Bethlenfalvay, G.L., R.L. Franson, M.S. Brown, and K.L. Mihara. 1989. The Glycine–Glomus–Bradyrhizobium symbiosis. IX. Nutritional, morphological and physiological responses of nodulated soybean to geographic isolates of the mycorrhizal fungus Glomus mosseae. Physiol. Plant. 76:226–232.
- Bray, R.H., and L.T. Kurtz. 1945. Determination of total, organic and available forms of phosphorus in soils. Soil Sci. 59:39–45.
- Byju, T. 2001. Soil analysis—A lab manual. Central Tuber Crop Research Inst., Sreekaryam, Trivandrum, India.
- Clark, R.B. 1997. Arbuscular mycorrhizal adaptation, spore germination, root colonization and host plant growth and mineral acquisition at low pH. Plant Soil 192:15–22.[CrossRef][Web of Science]
- Das, P.K., M.K. Sethi, Jena, and R.K. Patra. 1999. Effect of P sources and dual inoculation of VA mycorrhiza and Rhizobium on dry matter yield and nutrient uptake by green gram (Vigna radiata L.). J. Indian Soc. Soil Sci. 46:466–470.
- Diaza, G., P. Roldan, and J. Abbaladeje. 1992. Influence of soil type in colonization patterns and efficiency of mycorrhizal symbiosis of six Glomus sp. Mycologia 13(1):47–56.
- Douds, D.D., Jr., and N.C. Schenck. 1990. Relationship of colonization and sporulation by VAM fungus to plant nutrient and carbohydrate content. New Phytol. 116:621–627.[CrossRef][Web of Science]
- Gerdemann, J.W., and T.H. Nicolson. 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Br. Mycol. Soc. 46(2):235–244.
- Green, N.E., and S.O. Graham. 1976. The influence of the pH on the germination of Vesicular Arbuscular Mycorrhizal spores. Mycologia 68:929–934.[Web of Science]
- Hall, I.R., and B.J. Fish. 1979. A key to the Endogonaceae. Trans. Br. Mycol. Soc. 73(2):261–270.
- Hasan, A. 2002. Arbuscular mycorrhizal root colonization of some pulses in 7 districts of Eastern Uttar Pradesh—A preliminary report. Mycorrhiza News 14(3):15–16.
- Hayman, D.S. 1970. Endogone spore numbers in soil and vesicular arbuscular mycorrhizae in as influenced by season and soil treatment. Trans. Br. Mycol. Soc. 54(1):53–63.
- Hayman, D.S. 1983. The physiology of vesicular arbuscular mycorrhizal symbiosis. Can. J. Bot. 61:944–1024.
- Hazarika, D.K., K.K. Das, L.N. Dubey, and A.K. Phookan. 2000. Effect of Vesi. Arbus. Mycorrhizal (VAM) fungi and Rhizobium on growth and yield of green gram (Vigna radiata (L.) Wilczek.). J. Mycol. Plant Pathol. 30:424–426.
- Ikram, A. 1990. Beneficial soil microbes and crop productivity. Planter 66:777, 640–648.
- Jackson, M.L. 1962. Soil chemical analysis. Asia Publishing House, New Delhi, India.
- Jansa, J., A. Mozafar, T. Anken, R. Ruh, I.R. Sanders, and E. Frossard. 2002. Diversity and structure of AMF communities as affected by tillage in a temperate soil. Mycorrhiza 12:225–234.[CrossRef][Web of Science][Medline]
- Land, S., and F. Schonbeck. 1991. Influence of different soil types on abundance and seasonal dynamics of VAM fungi in the arable soils of N. Germany. Mycorrhiza 1:39–44.
- Land, S., H. von Alten, and F. Schonbeck. 1993. The influence of host plant, nitrogen fertilization and fungicide application on the abundance and seasonal dynamics of vesicular arbuscular mycorrhizal fungi in arable soils of N. Germany. Mycorrhiza 2(4):157–166.
- Marschner, H., and B. Dell. 1994. Nutrient uptake in mycorrhizal symbiosis. Plant Soil 159:89–102.[Web of Science]
- Menge, J.A. 1983. Utilization of vesicular-arbuscular mycorrhizal fungi in Agriculture. Can. J. Bot. 61:1015–1024.
- Muhr, C.R., N.P. Datta, H. Sankarasubramoney, V.K. Leley, and R.L. Donahue. 1965. Soil testing in India. USAID, New Delhi, India.
- Philips, J.M., and D.S. Hayman. 1970. Improved procedure for clearing roots and staining parasitic VAM for rapid assessment of infection. Trans. Br. Mycol. Soc. 55(1):158–161.
- Rao, V., and A.S. Rao. 1996. Response of blackgram (P. mungo) and green gram (P. radiatus) to inoculation with VAMF under different sources of Phosphorus fertilizer. Indian J. Agric. Sci. 66(10):613–616.
- Sidhu, O.P., and H.M. Behl. 1997. Response of three Glomus species on growth of Prosopis juliflora Swartz. at high pH levels. Symbiosis 23(1):23–34.[Web of Science]
- Smith, G.W., and H.D. Skipper. 1979. Comparison of methods to extract spores of vesicular arbuscular mycorrhizal fungi. Soil Sci. Soc. Am. Jr. 43:722–725.
- Sparks, D.L. 1996. Methods of soil analysis: Part 3—Chemical methods. SSSA Book Ser. No. 5. SSSA and ASA, Madison, WI.
- Sugavanam, V., K. Udaiyan, and S. Manian. 1994. Effect of fungicides on vesicular arbuscular mycorrhizal infection and nodulation in ground nut (Arachis hypogaea L.). Agric. Ecosyst. Environ. 48(3):285–293.
- Sullia, S.B., J. Prakash, and R.L.M. Pierik. 1991. Use of vesicular arbuscular mycorrhiza (VAM) as biofertilizer for horticultural plants in developing countries. Curr. Plant Sci. Biotechnol. Agric. 12:49–53.
- Sylvia, D.M., and L.H. Neal. 1990. Plant nitrogen stress affects the resistance of the host root to colonization by VAM fungi. New Phytol. 115(2):303–310.[Web of Science]
- Terry, E., Z. Teran, R. Martinez-Viera, and A. Pino-M-de-los. 2002. Biofertilizers, a promising horticultural production alternative in Organoponics. Cultivos Tropicales 23(3):43–46.
- Thakur, A.K., and J.D.S. Panwar. 1997. Response of Rhizobium—Vesicular Arbuscular. Mycorrhizal symbiosis on photosynthesis, nitrogen metabolism and sucrose translocation in green gram. Ind. J. Agric. Sci. 67(6):245–248.
TOP
ABSTRACT
INTRODUCTION
