Mycosphere

Compatible arbuscular mycorrhizal fungi of Jatropha curcas and spore multiplication using cereal crops

Charoenpakdee S1,2, Phosri C2, Dell B3, Choonluechanon S4 and Lumyong S1*
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200 Thailand Biology Program, Faculty of Science and Technology, Pibulsongkram Rajabhat University, Phitsanulok 65000, Thailand 3 Sustainable Ecosystems Research Institute, Murdoch University, Western Australia, 6150 Australia 4 Department of Soil Science and Conservation, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
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Charoenpakdee S, Phosri C, Dell B, Choonluechanon S, Lumyong S 2010 Compatible arbuscular mycorrhizal fungi of Jatropha curcas and spore multiplication using cereal crops. Mycosphere 1(3) 195204. Jatropha curcas is being considered as a biofuel crop for Thailand. Seedlings of J. curcas were used as bait plants to trap compatible arbuscular mycorrhizal fungi (AMF) in field soils in northern Thailand. Of the ten species of AMF that were trapped, two species, Scutellospora heterogama (CMU33) and Entrophospora colombiana (CMU05) produced abundant spores (>50 spores/100 g soil) and heavily colonized the roots of the trap plant. In a second experiment, the two AMF species were used to assess the effectiveness of four annual cereal crop plants (jobs tears, Coix lacrymajobi; rice, Oryza sativa; sorghum, Sorghum bicolor; maize, Zea mays) as suitable nurse plants for AMF spore multiplication. Higher mycorrhizal colonization and spore production were found after 120 days in sorghum than in the other crop species. Spore multiplication did not occur with corn and CMU33, nor with rice and CMU05. Except for the shoots of rice, inoculation increased the root and shoot dry weight of all four crop species. Sorghum is a suitable host for spore multiplication of E. colombiana but an alternative host, with the potential to produce higher spore yields, is required for S. heterogama. Key words Entrophospora sp. host plant Scutellospora sp. spore production, spore trapping Article Information Received 20 August 2010 Accepted 2 September 2010 Published online 8 October 2010 *Corresponding author: Saisamorn Lumyong e-mail [email protected] [email protected] and reducing the impact of plant pathogens (Mukeji & Ciancio 2007). Most of the research has focused on food and forest crops, and less attention has been paid to oil-yielding plants such as fennel (Foeniculum vulgare Mill.) (Kapoor et al. 2004), oil plam (Elaeis guineensis Jacq.) (Corley & Tinker 2003, Phosri et al. 2010) and menthol mint (Mentha arvensis L.) (Gupta et al. 2002). Physic nut (Jatropha curcas L.) is a drought-tolerant 195

Introduction Arbuscular mycorrhizal fungi (AMF) are natural plant growth stimulants (Wood & Cummings 1992). Most terrestrial plants form mycorrhizas (Smith & Read 1997) and many mycorrhizae have been shown to enhance plant survival and fitness through mechanisms such as increasing water and nutrient uptake (Marschner & Dell 1994, Peterson et al. 2004, Pasqualini et al. 2007, Plassard & Dell 2010)

shrub that is now being cultivated in industrial plantations in many countries, for example, Brazil, India, Mexico, Nicaragua and Thailand (Foidl et al. 1996, Heller 1996, David et al. 2009, Prueksakorn et al. 2006). The seeds contain 3137% oil content suitable for biodiesel production (Heller 1996). Although it is often reported that physic nut can grow rapidly and produce commercial oil yields on poor agricultural lands (Openshaw 2000, Pramanik 2003, Rao et al. 2008, Narenda et al. 2009), growth is likely to be slow unless there is appropriate application of beneficial organisms and balanced fertilizer. In a previous study, we showed that there is a high diversity of AMF associated with field-grown plants in northern Thailand (Charoenpakdee et al. 2010). Before these fungi can be recommended for commercial application, compatible species must be identified and procedures for inoculum production developed (Silva et al. 2005, Feldmann & Shneider 2008, Feldmann et al. 2008). Therefore, this study has two objectives: firstly, to use physic nut seedlings to trap compatible AMF species, and secondly to determine whether cereal crop species are suitable for spore multiplication. Methods Source of AMF inocula and trap culture Physic nut was used as a bait plant to trap compatible AMF in field soils collected from the rhizosphere of physic nut at ten sites in north and north-eastern Thailand. Samples were stored over ice for transportation, then at 4C for 1 week. Five hundred gram of soil from each sample was placed in the middle of 1.5 kg sterilized substrate in black plastic pots (15 cm top diameter). The substrate was an infertile soil from Mae Hea Agricultural Research Station and Training Center mixed with river sand (2:1, w/w). The soil was air dried and passed through a 4 mm mesh sieve, and the substrate was sterilized twice in an autoclave at 121C/15 psi for 30 minutes. The substrate had the following analysis: available P 4.9 mg.kg-1, total N 0.06 g.100 g-1, extractable K 104.6 mg.kg-1, organic matter 1.55%, and pHH2O 6.1. Physic nut seeds were disinfected with 0.5% sodium hypochloride for 5 minutes and 196

washed with sterilized water. Seeds were planted in autoclaved river sand and were watered with sterilized water until seed germination (2 weeks). Two uniform seedlings were transplanted into each pot and there were 3 replicate pots per soil collection. Seedlings were thinned to one per pot after 2 weeks and grown for 90 days in a randomized complete block design. Tap water was used to irrigate seedlings and watering stopped 7 days before harvesting to allow the substrate to gradually dry out. After harvest, the soil was maintained at 4C except when it was transferred to room temperature for spore assessment. Wet sieving and sucrose centrifugation methods were used to extract spores (Brundrett et al. 1996). The percent root colonization method was determined using 10% (w/v) potassium hydroxide clearing and 0.05% trypan blue staining at 121C for 15 minutes. Thirty root segments (each about 1 cm long) were assessed using the intercept method under a compound microscope (Brundrett et al. 1996). The criteria of AMF species selection for the next experiment was based on the number of spore multiplications (mean >50 spores per 100 g soil) and colonization (mean >70% of 30 root segments). Spores were identified using spore morphology (Trapp & Schenck 1982, Charoenpakdee et al. 2010, International Culture Collection of Vesicular and Arbuscular Endomycorrhizal Fungi [http://invam.caf.wvu.edu/Myc_Info/Taxonom y/species.htm]). The AMF were allocated Chiang Mai University culture collection accession numbers (Table 1) (Charoenpakdee et al. 2010). AMF sporulation in the rhizosphere of cereals Treatment combinations were performed in a factorial of 4 host plants 3 AMF treatments 3 replicates laid out in a completely randomized block design. Seeds of corn (Zea mays L.), jobs tears (Coix lacrymajobi L.), rice (Oryza sativa L.) and sorghum (Sorghum bicolor L.) were obtained from the Faculty of Agriculture, Chiang Mai University. These species were chosen because they are easy to grow in containers, are not known to require specific AMF, and are reported to be good hosts and trap plants for AMF due to characteristics such as fast growth, abundant

Mycosphere Table 1 AMF species, spore density (SD) per 100 gram of soil and percent colonization (C) from trap culture with physic nut seedlings harvested at 90 days.
AMF Code* Species SD Site* CR1 CMU31 S. pellucida 32.7a** CR2 CMU14 A. lacunosa 21.4a CM1 CMU33 S. heterogama 578.0b CM2 CMU06 A. scrobiculata 22.0a CM3 CMU05 E. colombiana 523.9b CM4 CMU14 A. lacunosa 34.3a LO1 CMU03 A. tuberculata 64.4a LP1 CMU12 A. excavata 23.2a KK1 CMU23 Glomus sp.4 94.2a NK1 CMU02 A. foveata 58.7a

115.2a 224.6a 974.6b 149.8a 895.2b 2012.7a 304.6a 170.0a 258.0a 1614.4a C *Chiang Rai site1 (CR1), Chiang Rai site2 (CR2), Chiang Mai site1 (CM1), Chiang Mai site2 (CM2), Chiang Mai site3 (CM3), Chiang Mai site4 (CM4), Loei (LO1), Lamphun (LP1), Khon Kaen (KK1), Nong Khai (NK1). **The same letters in each row indicate that there are no significant differences at P 0.05 using Tukey test. Values are mean (n=3) standard deviation.

Table 2 Comparison of spore number before (B) and after (A) trapping with physic nut seedlings.
AMF SD Code* Species Site* CR1 B A 7 3 CMU31 S. pellucida CR2 B A 14 2 CMU14 A. lacunosa B 3 CM1 A 57 CMU33 B 5 CM2 A 2 CMU06 CM3 B A 74 52 CMU05 E. colombiana CM4 B A 31 3 CMU14 A. lacunosa LO1 B A 31 6 CMU03 A. tuberculata LP1 B A 6 2 CMU12 A. excavata KK1 B A 17 9 CMU23 Glomus sp.4 NK1 B A 73 5 CMU02 A. foveata

S. heterogama

A. scrobiculata

*Chiang Rai site1 (CR1), Chiang Rai site2 (CR2), Chiang Mai site1 (CM1), Chiang Mai site2 (CM2), Chiang Mai site3 (CM3), Chiang Mai site4 (CM4), Loei (LO1), Lamphun (LP1), Khon Kaen (KK1), Nong Khai (NK1). **The same letters in each row indicate that there are no significant differences at P 0.05 using Tukey test.

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Fig. 1 AMF species trapped with physic nut as the bait plant viewed under stereomicroscope, scanning electron microscope and compound microscope (left to right). (a, b, c) Spores of Scutellospora heterogama (CMU33) showing bulbous subtending hyphae (H) and germination shield (GS) and (d, e, f) spores of Entrophospora colombiana (CMU05) showing spore ornamentation and stained with Melzers reagent. fine and hairy roots and tolerance to adverse conditions (Simson & Daft 1990, Brundrett et al. 1996, Habte & Osorio 2001). One hundred healthy seeds of each plant species were disinfected with 0.05% sodium hypocloride (NaOCl) for 5 minute and washed in sterile water. Then, all seeds were grown in sterile coarse river sand. After 7 days, 108 healthy and uniform seedlings of each host plant were randomly selected and planted, three seedlings per pot in each treatment. Scutellospora heterogama (CMU33) and Entrophospora colombiana (CMU05) were chosen for testing because they produced abundant spores (>50 spores/100 g soil) and heavily colonized the roots of the trap plants. Fifty spores of each fungus, taken from the trap culture experiment, were placed on sterile filter paper (Whatman No.1) and placed below the seed in each pot. The spores were washed with sterilized water prior to placement on the filter paper. The control treatment received filter paper without spores. The pots were black plastic pots, 25 cm in top diameter containing 2.5 kg of autoclaved sandy soil (prepared as above). Modified Hoagland solution (Gambrog & Wetter 1975) was prepared with low phosphorus (0.01 M, pH 6.5) and 100 ml applied to each pot once 198 per week. The pots were watered every other day with filtered tap water from which chlorine was evaporated for 24 hours in a 100 liter black tank before use. The pots were maintained in a greenhouse with natural photoperiod, temperature range of 2538C, and relative humidity between 44 and 88%, which is in the range for the wet season in Thailand (MayJuly 2008). Watering was stopped 7 days prior to harvesting at 120 days (all host plants had mature grain), to allow the soil and plants to dry slowly. Spore density per gram soil, percent colonization, height, and dry weight of shoots and roots were determined. Statistical analysis The data in percentage were transformed into arcsin for analysis. Univariate analysis was employed for percentage of colonization and spore density. One-way analysis of variance (ANOVA) was used for height, and weight of fresh and dry shoots and roots. Tukeys post hoc multiple mean comparison test was used to test significant differences between treatments at P 0.05. All statistical analyses were performed with Statistical Package for Social Sciences version 11.5 (SPSS Inc., Wacker Drive, Chicago, IL, USA).

Mycosphere Results Physic nut seedlings as bait plants Ten compatible morphospecies of AMF were trapped from the rhizosphere soil of physic nut under our experimental conditions. Surprisingly, after 3 months, the bait plants only trapped one sporulating AMF species from each site sample as follows: Scutellospora pellucida (T.H. Nicolson & N.C. Schenck) C. Walker & F.E. Sanders CMU31 in Chiang Rai site1 (CR1), Acaulospora lacunosa J.B. Morton CMU14 in Chiang Rai site2 (CR2) and Chiang Mai site4 (CM4), S. heterogama CMU33 in Chiang Mai site1 (CM1), A. scrobiculata CMU06 in Chiang Mai site2 (CM2), Entrophospora colombiana CMU05 in Chiang Mai site3 (CM3), A. tuberculata CMU03 in Loei (LO1), A. excavata CMU12 in Lumphun (LP1), Glomus sp.4 CMU23 in Khon Kean (KK1) and A. foveata CMU02 in Nong Khai (NK1). The percent colonization and spore density data showed similar trends. Except for two species, spore numbers and root colonization were low (Table 1). Two AMF exceeded the criteria (multiple spores, high root colonization) set for selection for the second experiment. They were S. heterogama (CMU33) and E. colombiana (CMU05) obtained from CM1 and CM3 (Fig. 1). They had high sporulation of 57 and 52 spores per 100 gram soil, and colonization of 97.3% and 89.0%, respectively. Trapping other site samples resulted in few spores and low ( 30%) infection rates (Table 1). The comparison of spore numbers before and after baiting showed that most AMF were not able to increase their spore densities under the experimental conditions (Table 2). AMF sporulation in the rhizosphere of cereals Sorghum had a higher percent colonization (68%) and sporulation (175 spores per gram soil) for E. colombiana (CMU05) than other host plants, whereas rice gave higher percentage of colonization (68%) for S. heterogama (CMU33) but sporulation was very low. Percent colonization in sorghum was 64% and spore number averaged 26 spores per gram soil for S. heterogama (CMU33) (Tables 3, 4). Sorghum had the highest percent root colonization following by jobs tears, corn and rice (Table 3). Spore production of S. heterogama (CMU33) did not occur with corn and likewise E. colombiana (CMU05) did not multiply with rice. However, there was some colonization of roots in corn for E. colombiana (CMU05) and in rice for S. heterogama (CMU33). The effect of AMF species on the dry weight of shoots and roots and plant height is shown in Table 5. All the parameters for inoculated plants followed similar trends with values generally higher than the controls. Except for shoot dry weight of rice, E. colombiana (CMU05) and S. heterogama (CMU33) promoted the dry weight growth of host plants. Futhermore, E. colombiana (CMU05) increased the dry weight of sorghum more than S. heterogama (CMU33). Discussion Spore production using physic nut as the bait plant Several different methods have been used to propagate AMF, which are obligate symbionts and cannot complete their life cycle without a host plant (Corkidi et al. 2008). The most widely used is pot culture, where the fungi are usually maintained and multiplied in combination with suitable host plant roots (Ferguson & Woodhead 1982). Native AMF inocula, which include spores, hyphae and root fragments colonized by AMF, can be obtained and produced from field soil using the rhizosphere soil of target plant species (Corkidi et al. 2008). Physic nut was used as trapping or bait plant for compatible AMF species as this was the target plant species of interest. In an earlier study, 34 AMF morphospecies were recorded from the rhizosphere of physic nut (Charoenpakdee et al. 2010), but given that weeds may have been hosting some of these fungi, it was unsure how many species of AMF were associated with physic nut roots in the field. In the current study, after 3 months, only 9 AMF species were reported to be sporulating and one species dominated at each sampling site. Most AMF were not able to increase their spore densities under rhizosphere of host plant in our experimental conditions. It is likely that some of the spores in the field soil were not 199

Table 3 Percent root colonization of four crop species inoculated with Entrophospora colombiana (CMU05) or Scutellospora heterogama (CMU33).
Host plant Control CMU05 CMU33 Mean Corn 0 36.86ab* 54.0018.00 32.0018.33 Jobs tears 0 49.14bc 49.3314.05 65.336.11 Rice 0 31.14a 24.0012.00 68.003.06 Sorghum 0 56.57c 68.006.93 64.006.93 0a* 48.83b 52.50b Mean *The same letters in each column and row mean that there were no significant differences at P 0.05 using the Tukey test. Values are mean (n=3) standard deviation.

Table 4 Spore density in the rhizosphere of four crop species inoculated with Entrophospora colombiana (CMU05) or Scutellospora hetergama (CMU33).
Host plant Control CMU05 CMU33 Mean Corn 0 0 22.14a* 51.663.84 Jobs tears 0 7.98a 3.000.32 15.632.50 Rice 0 0 0.03a 0.070.03 Sorghum 0 86.29b 175.0254.29 26.331.25 0a* 105.10c 17.42b Mean *The same letters in each column and row mean that there were no significant differences at P 0.05 using the Tukey test. Values are mean (n=3) standard deviation.

Table 5 Effect of Entrophospora colombiana (CMU05) or Scutellospora heterogama (CMU33) on growth of four host plants.
Jobs tears Rice Sorghum Shoot dry weight (g) Control 44.72.2a 50.10.8a 50.70.5a 57.20.9a CMU05 48.20.2b 56.40.9b 51.50.5a 63.42.0c CMU33 48.91.3b 54.52.0b 52.30.8a 60.45.5b Root dry weight (g) Control 30.86.6a 48.01.4a 41.32.1a 60.30.3a CMU05 42.01.6b 52.80.5b 44.10.1b 66.40.6c CMU33 40.92.4b 53.61.2b 45.30.6b 63.42.1b Height (cm) Control 65.71.3a 64.60.8a 61.50.9a 65.10.7a CMU05 67.51.3a 66.01.5a 62.30.9a 67.32.0a CMU33 66.31.3a 66.02.6a 62.73.0a 65.90.8a *The same letters in each column mean that there were no significant differences at P 0.05 using Tukey test. Values are mean (n=3) standard deviation. Treatment Corn

viable or that some were not compatible with physic nut roots. In addition, a more aggressive AMF that germinates rapidly may dominate by speedily colonizing roots and sporulating, such as in the case of S. heterogama. Furthermore, there may be some specificity for AMF and plant species concerning AMF development and sporulation (Liu & Wang 2003). The mycorrhizal dependency of a host is genetically fixed and the degree of mycorrhizal dependency can be expressed at the level of an 200

individual, or as a gradient within the ecological niche and relevant environmental conditions of the host (Feldmann et al. 2008). Clearly, as shown in other studies (van der Heijden et al. 1998, Douds & Millner 1999, Cardoso & Kuyper 2006, Wang et al. 2009), the combination of a suitable environment and host allowed E. colombiana (CMU05) and S. heterogama (CMU33) to proliferate in the pot study. The preliminary data suggest that physic nut may only form functional associations with

Mycosphere a limited range of AMF taxa. However, there are other possible explanations for the above finding. It is possible that some AMF may take longer than 3 months to produce spores or are recalcitrant under the pot conditions of the experiment. Our experimental conditions for sporulation, such as use of modified Hoaglands nutrient solution at pH 6.5 with low phosphorus level, may have constrained the development of some fungi as most of the initial soil inocula had a low pH. Carrenho et al. (2001) reported that the number of spores produced was influenced by soil chemical and physical properties and sporulation of Glomus macrocarpum was reduced at pH below 5.0. In the future, monosporic cultures will be required to evaluate the true extent of any host specificity with AMF and physic nut. Spore multiplication The evaluation of suitable pot hosts for AMF propagation was examined because the host type is one of the most important factors in optimizing spore production and multiplication (Safi & Khan 1997, Ryan & Graham 2002). In this study there was no cross contamination of the AMF species in any treatments, including the controls in the greenhouse, probably largely due to protection from rain splash. Spores can disperse by rain and germinate under optimal humidity (Brundrett et al. 1996). The present data indicated that AMF species can differ in their ability to infect different host plants. Entrophospora sp. (CMU05) had low colonization in rice and jobs tears and Scutellospora sp. (CMU33) had low colonization in corn. Differences in spore production under the different plant species may be due to characteristics of the host plant and environment interactions (Smith & Read 1997, Mukerji et al. 2002, Ryan & Graham 2002). This study found that the best growth promoting AMF species for sorghum and rice were Entrophospora sp. (CMU05) and Scutellospora sp. (CMU33), respectively. For corn and jobs tears, both AMF species could be suitable because there was no significant difference in promoting growth by the two species when inoculated with either AMF, though growth of both species was greater than the controls. Sorghum was more compatible as a host plant for spore multiplication, and thus similar to Glomus intraradices (Dabire et al. 2007) because their roots are fast-growing with extensive root systems and they are tolerant to fluctuating environmental conditions. Sorghum is also easy to grow in many parts of the world. Therefore, it is a good host plant for AMF propagation (Brundrett et al. 1996, Habte & Osorio 2001). Inoculum production on a commercial scale has always used widespread host plants such as Allium cepa L., Cenchrus ciliaris L., Panicum maximum Jacq., Paspalum notatum Fluegg, Sorghum halepense, Trifolium subterraneum L. and Zea mays in which spores develop within 3-4 months (Chellappan et al. 2001, Tahat et al. 2008). Future research will focus on Scutellospora heterogama (CMU33) and Entrophospora colombiana (CMU05) to determine their effectiveness in promoting growth and oil yield of physic nut in the field. The fungi may also prove to be useful for the management of mixed plantings, including some cereal and perennial cops. Acknowledgements Grants from the Commission of Higher Education, Thailand Research Fund: DBG4980004, and Chiang Mai University Graduate School are appreciated. We thank Dr. Morakot Sukchotiratana, Mr Keegan Kennedy and Ms Amornrat Jaiyasean for their kind assistance. References Brundrett M, Bougher N, Dell B, Grove T, Malajczuk N. 1996 Working with Mycorrhizas in Forestry and Agriculture. ACIAR Monograph 32. Canberra, Australia. Cardoso IM, Kuyper, TW. 2006 Mycorrhizas and tropical soil fertility. Agriculture, Ecosystems and Environment 116, 7284. Carrenho R, Silva ES, Trufem SFB, Bononi VLR. 2001 Successive cultivation of maize and agricultural practices on root colonization, number of spores and species of arbuscular mycorrhizal fungi. Brazilian Journal of Microbiology 32, 262270. 201

Charoenpakdee S, Phosri C, Dell B, Lumyong S. 2010 The mycorrhizal status of indigenous arbuscular mycorrhizal fungi of physic nut (Jatropha curcas L.) in Thailand. Mycosphere 1, 167181. Chellappan P, Christy SAA, Mahadevan A. 2001 Multiplication of arbuscular mycorrhizal fungi on roots. In: Techniques in Mycorrhizal Studies. Kluwer (eds KG Mukerji, C Manoharachary, BP Chamola) Academic Publishers, Netherlands. Corkidi L, Evans M, Bohn J. 2008 An introduction to propagation of arbuscular mycorrhizal fungi in pot cultures for inoculation of native plant nursery stock. Native Plants Journal 9, 2938. Corley RHV, Tinker PB. 2003 The Oil Plams, 4th edn Blackwell Science Ltd, Oxford, UK. Dabire AP, Hien V, Kisa M, Bilog A, Sangare KS, Galiana A, Prin Y, Duponnois R. 2007 Responses of soil microbial catabolic diversity to arbuscular mycorrhizal inoculation and soil disinfection. Mycorrhiza 17, 537545. David ML, Joerg AP, Alberte B. 2009 Modeling the land requirements and potential productivity of sugarcane and Jatropha in Brazil and India using the LPJmL dynamic global vegetation model. Biomass Bioenergy 33, 10871095. Douds DDJr, Millner PD. 1999 Biodiversity of arbuscular mycorrhizal fungi in agroecosystems. Agriculture, Ecosystems and Environment 74, 7793. Feldmann F, Shneider C. 2008 How to produce arbuscular mycorrhizal inoculum with desired characteristics. In: Mycorrhiza. (eds F Feldmann, Y Kapulnik, J Baar) Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany. Feldmann F, Hutter I, Schneider C. 2008 Best Production Practice of Arbuscular Mycorrhizal Inoculum. Federal Research Centre for Agriculture and Forestry, Messeweg, Germany. Ferguson JJ, Woodhead SH. 1982 Production of endomycorrhizal inoculum. An increase and maintenance of vesiculararbuscular mycorrhizal fungi. In: Methods and Principles of Mycorrhizal 202

Research.(ed NC Schenck) APS Press, St. Paul, MN. Foidl N, Foidl G, Sanchez M, Mittelbach M, Hackel S. 1996 Jatropha curcas L. as a source for the production of biofuel in Nicaragua. Bioresources Technology 58, 7782. Gambrog OL, Wetter LR. 1975 Plant Tissue Culture Method. Prairie Regional Laboratory. National Research Council of Canada. Saskatoon, Saskatchewan, Canada.Gubitz GM, Mittelbach M, Trabi M. 1999 Exploitation of the tropical oil seed plant Jatropha curcas L. Bioresources Technology 67, 7382. Gupta ML, Prasad A, Ram M, Kumar S. 2002 Effect of the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum on the essential oil yield related characters and nutrient acquisition in the crops of different cultivars of menthol mint (Mentha arvensis) under field conditions. Bioresources Technology 81, 7779. Habte M, Osorio NW. 2001 Arbuscular Mycorrhizas: producing and applying arbuscular mycorrhizal inoculum. University of Hawaii at Manoa, College of Tropical Agriculture and Human Resources, Honolulu, Hawaii. Heller J. 1996 Physic Nut. Jatropha curcas L. Promoting the conservation and use of underutilized and neglected crop. 1. Institute of Plant Genetics and Crop Plant Research, Gatersleben / International Plant Genetics Resources Institute, Rome. Kapoor R, Giri B, Mukerji KG. 2004 Improved growth and essential oil yield and quality in Foeniculum vulgare Mill on mycorrhizal inoculation supplemented with P-fertilizer. Bioresource Technology 93, 307311. Liu R, Wang F. 2003 Selection of appropriate host plants used in trap culture of arbuscular mycorrhizal fungi. Mycorrhiza 13, 123127. Marschner H, Dell B. 1994 Nutrient uptake in mycorrhizal symbiosis. Plant and Soil 159, 89102. Mukerji KG, Ciancio A. 2007 Mycorrhizae in the integrated pest and disease. Section 2. In Management General Concepts in

Mycosphere integrated pest and disease management. Springer Netherlands, 245266. Mukerji KG, Manoharachary C, Chamola BP. 2002 Techniques in Mycorrhizal Studies, 1st edn. Kluwer Academic Publishers, London-Netherlands. Narendra KS, Ashwani K, Satyawati S, Naik SN. 2009. Interaction of Jatropha curcas plantation with Ecosystem. Proceedings of International Conference on Energy and Environment 19-21 March, 2009. Openshaw K. 2000 A review of Jatropha curcas: an oil plant of unfulfilled promise. Biomass and Bioenergy 19, 115. Pasqualini D, Uhlmann A, Strmer LS. 2007 Arbuscular mycorrhizal fungal communities influence growth and phosphorus concentration of woody plants species from the Atlantic rain forest in South Brazil. Forest Ecology and Management 245, 148155. Peterson RL, HB Massicotte, Melville LH. 2004 Mycorrhizas: anatomy and cell biology. National Research Council of Canada, Ottawa, Ontario, Canada. Phosri C, Rodriguez A, Sander IR, Jeffries P. 2010 The role of mycorrhizas in more sustainable oil plam cultivation. Agriculture, Ecosystems and Environment 135, 187193. Plassard C, Dell B. 2010 Phosphorus nutrition of mycorrhizal trees. Tree Physiology 30, 11291139 doi:10.1093/treephys/tpq063 Pramanik K. 2003 Properties and use of Jatropha curcas oil and diesel fuel blends in compression ignition engine. Renewable Energy 28, 239248. Prueksakorn K, Shabbir HG, Pomthong M, Sbastien B. 2006 Energy analysis of Jatropha plantation systems for biodiesel production in Thailand, Energy for Sustainable Development, The 2nd Joint International Conference on Sustainable Energy and Environment (SEE 2006), 2123 November 2006, Bangkok, Thailand. Rao GR, Korwar GR, Shanker AK, Ramakrishna YS. 2008 Genetic associations, variability and diversity in seed characters, growth, reproductive phenology and yield in Jatropha curcas (L.) accessions. Trees 22, 697709. Ryan MH, Graham JH. 2002 Is there a role for arbuscular mycorrhizal fungi in production agriculture? Plant and Soil 244, 263271. Safi S, Khan A. 1997 The effect of vesicular arbuscular mycorrhizal association on growth of cereals, effect of barley growth. Plant and Soil 47, 1726. Silva FSB, Yano-Melo AM, Brando JAC, Maia LC. 2005 Sporulation of arbuscular mycorrhizal fungi using TrisHCl buffer in addition to nutrient. Brazilian Journal of Microbiology 36, 327332. Simson D, Daft MJ. 1990 Interactions between water-stress and different mycorrhizal inocula on plant growth and mycorrhizal development in maize and sorghum. Plant and Soil 121, 179186. Smith SE, Read DJ. 1997 Mycorrhizal Symbiosis, Second edition, Academic Press. London. Tahat MM, Kamaruzaman S, Radziah O, Kadir J, Masdek HN. 2008 Plant host selectivity for multiplication of Glomus mosseae spore. International Journal of Botany 4, 466470. Trapp JM, Schenck NC. 1982 Taxonomy of the fungi forming endomycorrhizae, a vesicular-mycorrhizal fungi (Endogonales). In: Method and Principles of Mycorrhizal Research. (ed NC Schenck NC) American Phytopathological Society, St. Paul, Minn. van der Heijden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T, Wiemken A, Sanders IR. 1998 Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396, 6972. Wang MY, Hu LB, Wang WH, Liu ST, Li M, Lui RJ. 2009 - Influence of long-term fixed fertilization on diversity of arbuscular mycorrhizal fungi. Pedosphere 19, 663-672. Wood T, Cummings B. 1992 Biotechnology and the future of VAM commercialization. In: Mycorrhizae in Sustainable Agriculture. (ed GJ Bethlenfalvay, RG 203

Linderman.). American Society of Agronomy Special Publication 54, 468487.

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