Diversity of Arbuscular Mycorrhizal Fungi in Mimosa invisa and Effect of the Soil ph on the Symbiosis

Chiang Mai J. Sci. 2010; 37(3) 517 Chiang Mai J. Sci. 2010; 37(3) : 517-527 www.science.cmu.ac.th/journal-science/josci.html Contributed Paper Diversity of Arbuscular Mycorrhizal Fungi in Mimosa invisa and Effect of the Soil ph on the Symbiosis Saengdao Kittiworawat [a], Somchit Youpensuk*[a], and Benjavan Rerkasem [b] [a] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. [b] Department of Agronomy, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand. *Author for correspondence; e-mail: scboi027@chiangmai.ac.th Received: 5 February 2010 Accepted: 22 June 2010 ABSTRACT Mimosa invisa is widely distributed in Northern Thailand. M. invisa is a leguminous weed which is used as green manure for increasing soil fertility and organic matter. They are colonized by arbuscular mycorrhizal (AM) fungi which increase nutrient uptake by the plant. In addition, roots of M. invisa are nodulated with Rhizobium bacteria that fix N 2 from the atmosphere. The objectives of this research are (1) to study diversity of AM fungi in M. invisa in cultivated, uncultivated and seasonally wet areas in Chiang Mai province (2) to evaluate the effect of soil ph (4.0, 5.0 and 6.0) on AM spore production in two varieties of M. invisa (thornless and thorny mimosa) and (3) the effect of AM fungi on the host plant in a pot experiment. Twenty-three species of AM fungi were found in the root zones of M. invisa in the study areas. The highest spore density occurred in uncultivated areas, slightly lower in cultivated areas and lowest in seasonally wet areas. In the pot experiment, AM fungi significantly increased dry weight, nutrient contents (N, P and K) in shoot, root and N in nodule in all treatments. The soil ph had effect on root colonization and spore density. The optimal soil ph for root colonization and spore production of AM fungi was ph 5.0 associated with both varieties of M. invisa. Root colonization in thornless mimosa and thorny mimosa in the soil ph 5.0 was 88% and 80%, respectively. The highest spore density in soil ph 5.0 of thornless mimosa was 16.3 spores g -1 soil while thorny mimosa was 7.3 spores g -1 soil. Thornless mimosa had root colonization and spore density of AM fungi higher than thorny mimosa. Therefore, in addition to the ease in handling without the sharp little thorns, the thornless mimosa is more suitable for using as green manure and as natural inoculum of AM fungi in agriculture than thorny mimosa. Keywords : arbuscular mycorrhizal fungi, Mimosa invisa, soil ph, Rhizobium. 1. INTRODUCTION Green manuring is widely used to improve soil fertility, especially where the use of chemical fertilizers is not feasible or is not appropriate, such as in organic agriculture. Legumes are generally used as green manure crops, as they can fix N 2 from the air in association with nodule bacteria. Legume crops and forage species have been found to

518 Chiang Mai J. Sci. 2010; 37(3) fix large amounts of nitrogen (N) from the air [1]. Mimosa invisa is used by farmers in northern Thailand as a cover crop as well as green manure [2]. Arbuscular mycorrhizal fungi (AM fungi) are soil-born fungi that associate widely with plant roots. They form mutualistic symbiosis with about 95% of all species of the plant kingdom. They are obligate symbionts that can not be propagated on artificial media without a living host [3]. AM fungi enhance uptake of immobile nutrients, especially P, and several micronutrients to the host plants. The external hyphae of AM fungi contribute to soil aggregation and structural stability [4]. Although no clear specificity between plant species and AM fungi has been described, effect of AM symbiosis in enhancing growth of the host plants may vary with fungal species [5]. Furthermore, AM fungi have been found to protect plants against root pathogens and restore degraded soil [6]. AM fungi are affected by several factors including soil, host plant, environmental conditions and agricultural practices [7]. A diverse range of AM fungi has been found associated with Macaranga denticulata, a pioneer tree species of Northern Thailand [8]. M. invisa, a pioneer legume, is commonly found in abandoned land as well as a weed in agricultural land, along with its relative, the giant mimosa, M. pigra. Our preliminary examination found that the roots of M. invisa were well colonized by AM fungi. It is therefore likely that this legume may derive benefits from symbioses with both N 2 fixing bacteria as well as AM fungi. The objectives of this study are (1) to investigate abundance and diversity of AM fungi in the rhizosphere of M. invisa in cultivated, uncultivated and seasonally wet areas in Chiang Mai province, (2) to evaluate the effects of soil ph (4.0, 5.0 and 6.0) on AM spore production of two varities of M. invisa (thornless and thorny mimosa) and (3) the effect of AM fungi on growth of the host plant in a pot experiment. 2. MATERIALS AND METHODS 2.1 The Study Areas The study areas were cultivated areas, uncultivated areas and seasonally wet areas in Muang, Saraphee and Maerim District, Chiang Mai Province, northern Thailand. Most of the soil samples were sandy clay loam textured of the San Sai series. Soil and root samples (0-15 cm depth) were collected from the root zones of M. invisa during August to October in 2007. From each study site, eight soil and root samples were collected. The soil ph of cultivated, uncultivated and seasonally wet areas was 5.1-5.9, 5.4-6.8 and 6.0-6.4, respectively. In the cultivated areas, M. invisa plants were cultivated in the same time with maize, but in the uncultivated and seasonally wet areas, the plants grew naturally. 2.2 Evaluation of Arbuscular Mycorrhizal Colonization in Root of Mimosa Invisa Root samples of M. invisa were washed and cut into about 1 cm. The root samples were cleared in 10% KOH at 121 o C about 15 min and washed in a sieve under running water. Cleared roots were stained with 0.05% trypan blue in lactoglycerol at 121 o C for 15 min. Thirty pieces of stained roots from each sample were mounted on glass slides to evaluate root colonization by AM fungi [9]. 2.3 Determination of Arbuscular Mycorrhizal Fungal Spores Spores of AM fungi were separated from 50 g of each soil sample from the root zones of M. invisa by wet sieving and 50% sucrose centrifugation [9]. After centrifugation, spores in the supernatant were poured over the 53 μm sieve and washed with tap water to remove the sucrose before vacuum filtration on filter paper and kept in Petri dishes. Spores were counted under a

Chiang Mai J. Sci. 2010; 37(3) 519 stereomicroscope. Different types of spores were observed under a compound microscope. Identification of AM fungi was done according to morphological characteristics of published AM spore descriptions [10, 11]. 2.4 Rhizobium Inoculum Nodules from roots of M. invisa were soaked with 1% sodium hypochlorite for 3 minutes and washed 3 times in sterile distilled water. The nodules were crushed and streaked on yeast extract mannitol agar with Congo red and incubated at 30 o C about 2 days. Single colony of Rhizobium was transfered to yeast extract mannitol broth and cultivated in shaker at 30 o C [12]. 2.5 Pot Experiment for the Effect of Soil ph to AM Fungi Associated with M. Invisa The experiment was a full factorial with AM fungal inoculation (inoculated and non-inoculated treatments), three level of ph (4.0, 5.0 and 6.0) with four replications. Five kilograms of steam sterilized soil were used per pot in this experiment. The soil used in this experiment was sandy loam textured. It had a ph 6.0 and contained 0.04% N (Kjeldahl method), 124.12 mg.kg -1 soil available P (Bray II method) and 386.5 mg.kg -1 soil extractable K (1 N NH 4 OAc, ph 7). The soil was adjusted to ph 4.0 and 5.0 by mixing with Al 3 (SO 4 ) 2. 18H 2 O. Rhizobium inoculum (10 8 cfu/ml) was poured on roots of M. invisa before transplanting. About 250 spores of mixed species of AM fungi were inoculated in the bottom of the planting hole of inoculated treatments before transplanting. Four months after transplanting, leaf and root were harvested to determine root and shoot dry weight. Roots samples from inoculated treatment were determined root length colonization of AM fungi. The soil samples from inoculated treatments were determined spore densities of AM fungi. Leaves and stems from each treatment were evaluated for N (Kjedahl method) [13], P (dry ashing and molybdovanado-phosphoric acid) [14] and K (dry ashing, and atomic absorption spectrophotometer method) [13]. 2.6 Statistical Analysis Statistical tests were performed with SPSS version 14. The data were analyzed by analysis of variance (ANOVA) to test the effect of the factors. Mean comparisons were determined by Waller-Duncan at p < 0.05. 3. RESULTS AND DISCUSSION Twenty three species of AM fungi were found in rhizosphere of M. invisa in all the soil samples in the study sites (Table 1). The genera were identified as Acaulospora (7 species), Glomus (14 species), Gigaspora (1 specie) and Scutellospora (1 specie) (Table 2). Species richness of AM fungi was 16 species at cultivated areas, 18 species at seasonally wet areas and 18 species at uncultivated areas (Table 3). Root colonization in cultivated areas, uncultivated areas and seasonally wet areas were 90 %, 94% and 91%, respectively. The spore density was significantly different in uncultivated, cultivated and seasonally wet areas with 20.9, 12.5 and 9.8 spores g -1 soil, significantly (Figure 1). Uncultivated areas had spore density higher than cultivated areas because agricultural practices such as tillage, application of chemical fertilizer and pesticides have negative effects on the AM fungi. Tillage can reduce either AM spore density [15] or AM colonization [16]. In Maize, no tilled soil had higher density of AM hyphae than tilled soil because tillage damaged AM hyphae and diversity of AM fungi [17]. Diversity of AM fungi in the rhizosphere of M. invisa, Glomus etunicatum, Glomus geosporum, Glomus constrictum and Acaulospora scobiculata were frequently found in the study areas. Whereas Gigaspora margarita

520 Chiang Mai J. Sci. 2010; 37(3) Table 1. AM fungi associated with M. invisa at the study areas in Chiang Mai Province. Genera Acaulospora Glomus Gigaspora Scutellospora Species Acaulospora capsicula Acaulospora lacunose Acaulospora mellea Acaulospora rhemii Acaulospora rugosa Acaulospora scobiculata* Acaulospora spinosa Glomus ambisporum Glomus australe Glomus botryoides Glomus cavisporum Glomus clarum Glomus constrictum* Glomus dusii Glomus etunicatum* Glomus geosporum* Glomus macrocarpum Glomus mosseae Glomus multicaule Glomus rubiforme Glomus sinuosum Gigaspora margarita Scutellospora biornata * Frequently found in all study areas. was found in the cultivated and uncultivated areas, Scutellospora biornata was only found in the uncultivated areas. It was shown that properties of soil had effected on species of AM fungi. Diversity and number of spore of AM fungi may depend on environment factors. In the pot experiment, AM fungal inoculation increased total dry weights (Table 4) and nutrient contents (N, P and K) in shoots (Table 5), roots (Table 6) and N in nodules (Table 7) of M. invisa (thorny and thornless mimosa). However, the effect varied with the Table 2. AM fungi associated with M. invisa at the area of study in Chiang Mai Province. Relative Genera of Number of abundance AM fungi species of each genus Acaulospora 7 + + + + Gigaspora 1 + Glomus 14 + + + + Scutellospora 1 + Total 23 soil ph. Furthermore in this experiment, AM fungi increased N accumulation in root nodules. It indicated that AM fungi had positive association with Rhizobium in enhancing growth of the host plants. The total dry weights of inoculated plants were significantly higher than non-inoculated plants in all treatments. Groundnut inoculated with dual inoculation of AM fungi (Glomus mossae) and Rhizobium was reported to increase significantly more growth, number of root nodules, mycorrhizal colonization compared with those inoculated with either AM fungi or Rhizobium alone [18]. AM fungi in both varieties of M. invisa showed a response to the soil ph treatments (Table 8). Root colonization and spore density in inoculated treatments was highest in soil ph 5.0 in both plant varieties and lowest in soil ph 6.0 in thornless mimosa and in soil ph 4.0 in thorny mimosa. In soil ph 5.0, the highest root colonization of AM fungi in thornless mimosa and thorny mimosa was 88% and 80%, respectively, and highest spore densities of AM fungi in thornless mimosa was 16.3 spores g -1 soil of soil and thorny mimosa was 7.3 spores g -1 soil. Soil ph had influence on root colonization and spore density of AM fungi. Soil ph 5.0 was optimal for root colonization and spore production of AM fungi associated with M. invisa. But thornless mimosa had higher root

Chiang Mai J. Sci. 2010; 37(3) 521 Table 3. Numbers of species of AM fungi associated with M. invisa at each study areas in Chiang Mai Province. Genera of Cultivated Uncultivated Seasonally AM fungi areas areas wet areas Acaulospora 5 4 5 Glomus 10 11 12 Gigaspora 1 1 - Scutellospora - 1 - Total 16 17 17 Figure 1. (A) Percentage of root colonization in rhizoshere of M. invisa and (B) Spore density of AM fungi in cultivated areas, uncultivated areas and seasonally wet areas. Means with different letters were significantly different by Waller-Duncan at p < 0.05. The error bars were calculated from standard deviation.

522 Chiang Mai J. Sci. 2010; 37(3) Table 4. Effects of inoculation and soil ph on shoot and root dry weight of four-month M. invisa seedlings. Shoot dry Root dry Total dry Type of mimosa Soil ph Inoculation weigth weigth weigth (g/plant) (g/plant) (g/plant) Thornless mimosa 4 Non-AM 17.52d 5.11abc 22.63d 4 AM 19.86cd 6.38a 26.25cd Thornless mimosa 5 Non-AM 22.17cd 3.82cd 26.03cd 5 AM 28.81b 6.41a 35.25b Thornless mimosa 6 Non-AM 20.05cd 3.81cd 23.90d 6 AM 27.94b 5.23ab 33.18bc Thorny mimosa 4 Non-AM 20.41cd 4.65bc 25.05d 4 AM 20.65cd 5.27ab 25.95d Thorny mimosa 5 Non-AM 22.48cd 3.82cd 26.33cd 5 AM 38.61a 4.54bc 43.15a Thorny mimosa 6 Non-AM 19.86cd 2.80d 22.65d 6 AM 22.67c 3.05d 25.73d Analysis of variance Type of mimosa ns * ns Inoculation * * * Soil ph * * * Type of mimosa x Inoculation ns * ns Type of mimosa x ph * ns * Inoculation x ph * ns * Type of mimosa x Inoculation x ph * ns * AM, inoculated; Non-AM, non-inoculated with AM fungi. Means in the same column followed by different letters are significantly different by Waller-Duncan. * = significant at P < 0.05, ns = not significant. colonization and spore density than thorny mimosa. Therefore, thornless mimosa is more suitable for using as green manure in acidic soil than thorny mimosa. Thornless mimosa also produced twice as many spores than thorny mimosa in soil with ph 4.0-5.0, which means that it would be a better source of AM inoculant. 4. CONCLUSION Twenty-three species of AM fungi were found from the rhizosphere of M. invisa. They were placed in four genera: Acaulospora (7 species), Gigaspora (1 species), Glomus (14 species) and Scutellospora (1 species). Land use history had only a small effect on diversity of AM fungi. AM fungi significantly increased

Chiang Mai J. Sci. 2010; 37(3) 523 Table 5. Effects of inoculation and soil ph on shoot N, P and K contents of four-month M. invisa seedlings. Type of mimosa Soil ph Inoculation Shoot nutrient content N (%) P (%) K (%) Thornless mimosa 4 Non-AM 1.79efg 0.10f 1.06e 4 AM 2.02bc 0.11ef 1.63bc Thornless mimosa 5 Non-AM 1.82def 0.12ef 1.12e 5 AM 2.30a 0.20ab 1.70abc Thornless mimosa 6 Non-AM 1.67fg 0.17bcd 1.25de 6 AM 2.13ab 0.22a 2.05a Thorny mimosa 4 Non-AM 1.64fg 0.10f 1.06e 4 AM 1.92cde 0.13def 1.51cd Thorny mimosa 5 Non-AM 1.60g 0.15cde 1.41cde 5 AM 2.05bc 0.20ab 1.91ab Thorny mimosa 6 Non-AM 1.39h 0.10f 1.24de 6 AM 1.82def 0.18abc 1.94ab Analysis of variance Type of mimosa * ns ns Inoculation * * * Soil ph * * * Type of mimosa x Inoculation ns ns ns Type of mimosa x ph ns * ns Inoculation x ph ns ns ns Type of mimosa x Inoculation x ph ns ns ns AM, inoculated; Non-AM, non-inoculated with AM fungi. Means in the same column followed by different letters are significantly different by Waller- Duncan. * = significant at P<0.05, ns = not significant. growth and nutrient uptake in M. invisa. The soil ph 5.0 gave the highest root colonization and spore production of AM fungi associated with M. invisa (thorny and thornless mimosa). Thornless mimosa is more suitable than thorny mimosa for using as green manure and as production of natural AM fungi inoculum, especially in acidic soils. 5. ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support from the Thailand Research Fund and the Mcknight foundation.

524 Chiang Mai J. Sci. 2010; 37(3) Table 6. Effects of inoculation and soil ph on root N, P and K contents of four-month M. invisa seedlings. Type of mimosa Soil ph Inoculation Shoot nutrient content N (%) P (%) K (%) Thornless mimosa 4 Non-AM 1.01ef 0.05f 1.31c 4 AM 1.32abcd 0.07def 1.95a Thornless mimosa 5 Non-AM 1.17cde 0.09d 1.49bc 5 AM 1.38abc 0.12abc 2.08a Thornless mimosa 6 Non-AM 1.19bcde 0.09cd 1.43bc 6 AM 1.49a 0.13a 1.89a Thorny mimosa 4 Non-AM 0.94f 0.06ef 1.37c 4 AM 1.15def 0.09de 1.83ab Thorny mimosa 5 Non-AM 0.97ef 0.09bcd 1.40c 5 AM 1.35abcd 0.12abc 2.12a Thorny mimosa 6 Non-AM 1.00ef 0.08d 1.19c 6 AM 1.41ab 0.12a 2.10a Analysis of variance Type of mimosa * ns ns Inoculation * * * Soil ph * * ns Type of mimosa x Inoculation ns ns ns Type of mimosa x ph ns ns ns Inoculation x ph ns ns ns Type of mimosa x Inoculation x ph ns ns ns AM, inoculated; Non-AM, non-inoculated with AM fungi. Means in the same column followed by different letters are significantly different by Waller-Duncan. * = significant at P<0.05, ns = not significant.

Chiang Mai J. Sci. 2010; 37(3) 525 Table 7. Effects of inoculation and soil ph on nodule N contents of four month M. invisa seedlings. Type of mimosa Soil ph Inoculation N content in Number of nodule (%) nodule Thornless mimosa 4 Non-AM 2.90e 799b 4 AM 3.56d 455e Thornless mimosa 5 Non-AM 4.13c 930a 5 AM 4.33abc 212g Thornless mimosa 6 Non-AM 3.71d 345f 6 AM 4.42ab 223g Thorny mimosa 4 Non-AM 3.68d 734c 4 AM 4.18bc 629d Thorny mimosa 5 Non-AM 3.80d 799b 5 AM 4.42ab 182g Thorny mimosa 6 Non-AM 4.10c 455e 6 AM 4.48a 225g Analysis of variance Type of mimosa * ns Inoculation * * Soil ph * * Type of mimosa x Inoculation ns * Type of mimosa x ph * * Inoculation x ph * * Type of mimosa x Inoculation x ph ns * AM, inoculated; Non-AM, non-inoculated with AM fungi. Means in the same column followed by different letters are significantly different by Waller-Duncan. * = significant at P<0.05, ns = not significant.

526 Chiang Mai J. Sci. 2010; 37(3) Table 8. Means of root colonization and spore density in roots of M. invisa at different soil ph. Treatment Soil ph AM colonization Spore density (%) (spores g -1 soil) Thornless mimosa 4 59.00c 9.6b Thorny mimosa 4 47.50d 4.1e Thornless mimosa 5 87.75a 16.3a Thorny mimosa 5 80.75a 7.3c Thornless mimosa 6 49.75d 5.8d Thorny mimosa 6 69.75b 4.3e Analysis of variance Type of mimosa ns * Soil ph * * Type of mimosa x soil ph * * Means in the same column followed by different letters are significantly different by Waller-Duncan. * = significant at P < 0.05, ns = not significant. REFERENCES [1] Peoples M.B. and Herridge D.F., Nitrogen fixation by legumes in tropical and subtropical agriculture, Adv. Agron., 1990; 44: 155-223. [2] Rerkasem K. and Pinedo-Vasquez P., Diversity in Smallholder Systems and Responses to Environmental and Economic Changes; in Jarvis D., Padoch C., and Cooper H. D., eds., Managing Biodiversity in Agricultural Ecosystems. Columbia University Press, 2007: 362-381. [3] Sieverding E., Vesicular-Arbuscular Mycorrhiza Management in tropical Agrosystems, Technical Cooperation, Federal Republic of Germany, Eschborn, 1991. [4] Cardoso I.M. and Kuyper T.W., Mycorrhizas and tropical soil fertility, Agr. Ecosyst. Environ., 2006; 116: 72-84. [5] Youpensuk S., Rerkasem B., Dell B. and Lumyoung S., Effects of arbuscular mycorrhizal fungi fallow enriching tree (Macaranga denticulate), Fungal Divers., 2005; 18: 189-199. [6] Quilambo O.A., The vesicular-arbuscular mycorrhizal symbiosis, Afr. J. Biotechnol., 2003; 2: 539-546. [7] Hayman D.S., Influence of soils and fertility on activity and survival of Vesicular arbuscular mycorrhizal fungi, Phytopathol., 1982; 72: 1119-1125. [8] Youpensuk S., Lumyoung S., Dell B. and Rerkasem B., Arbuscular mycorrhizal fungi in the rhizosphere of Macaranga denticulate Muell. Arg., and their effect on the host plant, Agrofor Syst., 2004; 60: 239-246. [9] Brundrett M., Bougher N., Dell B., Grove T. and Malajczuk N., Working with mycorrhizas in forestry and agriculture, ACIAR Monograph, Canberra, 1996. [10] Schenck N.C. and Perez Y., Manual for the Identification of VA Mycorrhizal Fungi, 2 nd Edn., INVAM Gainesville, Florida, USA, 1988.

Chiang Mai J. Sci. 2010; 37(3) 527 [11] INVAM website (2005) http://invam. caf.wvu.edu/fungi/taxonomy/classification.htm. [12] Vincent J.M., A Manual for the Practical Study of Root Nodule Bacteria, Blackwell Scientific, Oxford, 1970. [13] Jackson H., Soil Chemistry Analysis, Prentice-Hall of India Private Limited, New Delhi, 1967. [14] Delhaize E., Dell B., Kirk G., Loneragan J., Nable R., Plaskett D. and Webb M., Manual of research procedure: plant nutrition research group school of environmental and life sciences, 1 st Edn., Murdoch University, Perth, Australia, 1984. [15] Kabir Z., O Halloran I.P., Widden P. and Hamel C., Vertical distribution of arbuscular mycorrhizal fungi under corn (Zea mays L.) in no-till and conventional tillage systems, Mycorrhiza, 1998; 8: 53-55. [16] McGonigle T.P., Evans D.G. and Miller M.H., Effect of degree of soil disturbance on mycorrhizal colonization and phosphorus absorption by maize in growth chamber and field experiments, New Phytol., 1990; 116: 629-636. [17] Kabir Z., O Halloran I.P., Fyles J.W. and Hamel C., Seasonal changes of arbuscular mycorrhizal fungi as affected by tillage practices and fertilization: I. hyphal density and mycorrhizal root colonization, Plant Soil, 1997; 192: 285 293. [18] Devi C.M. and Reddy N.M., Growth response of ground nut to VAM fungus and Rhizobium inoculation, Plant Pathol. Bull., 2001; 10: 71-78.