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Propagation technique of arbuscular mycorrhizal fungi isolated from coastal reclamation land
European Journal of Soil Biology 74 (2016) 39e44
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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Propagation technique of arbuscular mycorrhizal fungi isolated from coastal reclamation land Gopal Selvakumar, Ramasamy Krishnamoorthy 1, Kiyoon Kim, Tongmin Sa* Department of Environmental and Biological Chemistry, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 November 2015 Received in revised form 2 March 2016 Accepted 14 March 2016
Arbuscular mycorrhizal fungi (AMF) are well known for their plant growth promoting potential and stress tolerance ability. AMF promotes plant growth even at adverse environmental conditions and improve plant nutrient uptake. This study aimed to propagate AMF using a single spore inoculation technique. Soil samples were collected from salt affected Saemangeum reclaimed land. Collected soil samples were subjected to soil analysis. In the single spore inoculation method, sorghum sudangrass (Sorghum bicolor L.) was inoculated with a single spore. Among the 150 inoculants, six spores were able to germinate in vitro. Geminated spores were transferred to 1 kg pots containing sterilized field soil. After 120 days, the contents were mixed and transferred to 2.5 kg pots and maintained for another 120 days. After 240 days, spore count and colonization were checked. The propagated spores were identified using nested PCR followed by sequencing. The 18S rDNA sequencing of spores revealed that the spores belonged to Gigaspora margarita and Claroideoglomus lamellosum. Among the 6 inoculants, Claroideoglomus lamellosum S-11 (391 spore per 100 g of soil) had the highest spore count followed by Gigaspora margarita S-23 (235 spore per 100 g of soil). Slide method allowed visual monitoring of spore germination in vitro as well as being able to mass produce pure cultures of AMF for bio-inoculation purposes. © 2016 Elsevier Masson SAS. All rights reserved.
Handling Editor: Prof. C.C. Tebbe Keywords: Mass production Arbuscular mycorrhizal fungi Slide method Gigaspora margarita Claroideoglomus lamellosum
1. Introduction Arbuscular mycorrhizal fungi (AMF) are widespread soil fungi which form mutualistic symbiosis with more than 80% of land plants and are believed to be obligate symbiotic biotrophs. Only the intraradical hypha of this fungus takes up hexose as carbon source in the apoplast of the plant root cortex cells to complete their life cycle and sporulate [1]. Therefore, the ability of AMF to utilize externally supplied carbon source in asymbiotic medium is limited. Although AMF are indigenous to agricultural soils, inoculation of these fungi improves plant growth by enhancing the uptake of immobile soil mineral nutrients and water [2]. AMF have increased the growth of various field-grown crops, including cotton, tomato, orange, pepper and onions [3]. In addition to enhancement of
* Corresponding author. Department of Environmental and Biological Chemistry, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea. E-mail address: [email protected] (T. Sa). 1 Present address: Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai, India. http://dx.doi.org/10.1016/j.ejsobi.2016.03.005 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
nutrient uptake by plants, AMF also help plants withstand various biotic and abiotic stresses such as pathogen attack [4], drought stress [5] and salt stress [6]. Enhanced plant growth through AMF inoculation can be ensured by two principal ways (1) selection and inoculation of efficient mycorrhizal fungi and (2) promotion of the efficient native mycorrhizal fungi [7]. Although previous reports support that some AMF are beneficial to a wide variety of plant species [8], some reports argue that host specificity of AMF might reduce the mycorrhizal interaction between plant and fungi [9,10]. AMF inocula that are commercially available come in a variety of forms at low to high concentration and from single inoculum to multiple combined inoculum in a carrier material. However, their performance in nonindigenous soil tends to decrease due to their lack of host specificity and differences in agricultural management practices [11]. Estrada et al. [12] reported that native AMF species from Mediterranean saline soil are more effective in improving plant growth than the introduced AMF species. In addition, reintroduction of native AMF inocula may be necessary to overcome the harmful effects of previous agricultural management, e.g. excessive use of fungicide. Purchasing large amounts of inoculum necessary for large-scale
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agriculture is not cost-effective. Moreover, even though inocula of AMF are commercially available, production of pure cultures of host specific AMF is essential. Development of pure culture also enables the study of the morphological characteristics of new AMF species and their individual effect on plant growth. Previously, pure cultures of AMF have been obtained in vitro using Ri-TDNA transformed root organ culture [13,14], aeroponics [15,16] and bicompartmental Petri plates [17]. However, these in vitro methods limit their application in the field due to their requirement of precolonized plants and different growth medium [18]. Although pot culture and monoxenic culture propagated spores share similar ontogeny, daughter spores from monoxenic cultures usually differentiate into smaller spores with paler pigmentation and thinner laminated spore walls [19,20]. The inocula produced by substrate-free cultivation method may require carrier material before their application in large scale to improve their survivability. In addition, inocula produced through soil-based method are least artificial, most adopted and can be applied directly in the field [11]. The present study aimed to propagate the AMF spores isolated from Saemangeum reclaimed land using single spores as starter inoculum. 2. Materials and methods 2.1. Study area and soil sample collection Saemangeum is one of the world's largest reclamation sites adding about 400 km2 to South Korea's total geographical area. A total of thirty five rhizosphere soil samples (10 cm radius and 15 cm depth; approximately, one kg for each sample) have been collected from Saemangeum reclamation land. The distance between each sample was at least 10 m, so that the chances of sharing the same AMF species between samples are reduced. Based on the initial spore count, ten soil samples which had over 10 healthy spores were used in this study. Soils from rhizospheres of dominant plant species such as Phragmites australis, Cyperus polystachyos and Miscanthus sinensis were chosen to obtain different AMF isolates. Each rhizosphere soil sample along with the plant roots was collected in a sterilized polyvinyl chloride bag and transported immediately to the laboratory in icebox and kept at 4 C until use. 2.2. Soil analysis and initial spore enumeration All the collected soil samples were subjected to initial spore count and determination of electrical conductivity (EC) value (1:5 dilution method). The initial spore count of the soil samples were assessed using wet sieving and decanting method as described by Daniels and Skipper [21] followed by sucrose centrifugation method. Soil properties such as pH, cation exchange capacity (CEC), organic matter, micro and macro nutrient contents were examined. 2.3. Single spore - slide method Freshly isolated spores were examined under the microscope and only the healthy and damage-free spores were used for propagating the AMF in vitro. Slide method was conducted to visually monitor spore germination. Briefly, sterilized filter paper (Whatman No. 6) was excised to ¾ of the glass slide and tied with rubber band and kept in a 50 ml sterile falcon tube containing the mixture of plant root exudates and sterilized Hoagland's nutrient solution. The bottom of the filter paper was in contact with the solution so that the moisture can be maintained throughout the experiment. The plant root exudates were obtained as described in Panwar et al. [22]. Sorghum sudangrass seeds were surface sterilized by immersing them in 70% ethanol for 2 min followed by 1% sodium
hypochlorite (NaOCl) for 3 min and thoroughly rinsed with sterile distilled water for seven to ten times. The surface sterilized seeds were placed in a petri dish containing sterile moist filter paper and kept in the growth chamber for 2e3 days. The growth chamber conditions were as follows: 12 h of light at 25 C and 12 h of dark at 20 C and maintenance of 70% humidity. The germinated seeds were transferred to slides and tied with rubber band without harming the radicles. A healthy single spore was placed near the growing root (Fig. 1). A total of 150 inoculants were made. The setup was kept in the growth chamber with the above mentioned conditions for two weeks. Every 3 days the spore was examined under the microscope for germination. After 2 weeks, six slides with successfully germinated spores were transferred to 1 kg pots containing sterilized soil without disturbing the setup and new pregerminated sorghum seeds were sown. Sterilized field soil consecutively autoclaved for three days was added to fill up the remaining space in the pots. New pre-germinated sorghum seeds were sown and the plants were allowed to grow for 120 days. Hoagland's nutrient solution was poured on every pot every week. After 105 days, the plants were subjected to drought stress by withholding water or Hoagland's nutrient solution for 15 days to induce spore production. After 120 days, the soil from each pot was mixed and transferred to 2.5 kg pot with sterilized soil. Pre-germinated sorghum seeds were sown and grown for another 120 days following the above mentioned procedure. After 240 days, root and soil samples were collected and examined for mycorrhizal colonization and spore numbers. Briefly, the roots were first washed with 10% KOH for 10 min in water bath at 90 C. Then the roots were washed with tap water and immersed in 2% HCl for 10 min at room temperature. After discarding the HCl, 0.5% trypan blue in lactoglycerol was added and the roots were allowed to stain at 90 C for 10 min. The staining solution was discarded and the stained roots were washed with tap water. Roots were immersed in destaining solution for overnight to remove excess staining and were checked for colonization [23]. The stained root fragments (1 cm) were arranged in glass slides and observed under the microscope for the presence of hypha, vesicles and arbuscules. Scoring was done based on the intensity of colonization (0e5) and based on the arbuscules intensity (A0eA3) as described by Trouvelot et al. [24]. A total of 30 root fragments were observed for each treatment. Intensity of the mycorrhizal root colonization was estimated as the amount of cortex cell that colonized by mycorrhiza relative to the whole root system (M%). Abundance of arbuscule was estimated as the arbuscule richness in the whole root system (A%). The Mycocalc software was used to determine the M%, and A%. 2.4. 18S rDNA sequencing Five healthy spores from successfully propagated slide method pots were taken in microcentrifuge tube and surface sterilized with 2% chloramine-T and 100 mg/ml streptomycin for 30 min. Then the spores were transferred to a sterilized PCR tube containing 10 ml of 1:1 ratio of 10X PCR buffer and sterilized distilled water. Spores were aseptically crushed with a sterilized blunt end pasteur pipette. The 18S rDNA of arbuscular mycorrhizal fungal spores were amplified using the nested PCR [25]. In the first round of PCR, eukaryotic genes were targeted using universal eukaryotic primers GeoA2 (50 -CCAGTAGTCATATGCTTGTCTC-30 ) and Geo11 (50 ACCTTGTTACGACTTTTACTTCC-30 ) which amplified the genes with the product length of 1800 bp. The final product was diluted 1:100 ratio with TE buffer [26] and used as template for next PCR. In the second round of PCR, AMF specific primer AM1 (50 GTTTCCCGTAAGGCGCCGAA-30 ) was used to amplify AMF specific
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Fig. 1. Single spore inoculation, slide method. (a) aseptically germinated seedling tied on a glass slide covered with sterilized filter paper secured, (b) slide kept in falcon tube containing root exudates, (c) freshly isolated AMF spore inoculated near growing root of sorghum Sudangrass. SS e Sorghum-sudan grass seedling, RE e root exudate, R e root and S e spore.
region along with NS31 (50 -TTGGAGGGCAAGTCTGGTGCC-30 ) targeting the product size of 550 bp. Nearly complete 18S rDNA sequences were aligned and the closest fungal identities were obtained after deduced by BLAST search. The phylogenetic tree was generated after multiple alignment with CLUSTAL-W [27] and the tree topologies were evaluated by bootstrap analysis of 1000 dataset using MEGA version 6.0 software [28]. The nucleotide sequence of 18S rDNA sequences were deposited in GenBank and the accession numbers were given as KP677594 to KP677599.
these inoculants propagated over 200 spores per 100 g of soil (Table 2). However, the highest mycorrhizal colonization was observed in Gigaspora margarita S-9 followed by Gigaspora margarita S-23. The highest arbuscule abundance was observed in Gigaspora margarita S-20 followed by Gigaspora margarita S-9. 3.3. 18S rDNA sequencing Six inoculants were taken for further identification by amplifying the 18S region of fungal DNA and their closest neighbors were identified (Fig. 3). Five of the isolates belong to Gigaspora margarita and one isolate belong to Claroideoglomus lamellosum.
3. Results 3.1. Soil analysis and initial spore enumeration
4. Discussion
The EC value of the Saemangeum soil samples varied from 0.30 to 1.60 dS/m. Most of the spores were observed to be damaged probably due to the salinity level of Saemangeum soil. The soil physico-chemical properties show that nitrogen, phosphorous and calcium contents of Saemangeum reclaimed soil is very low compared to normal field soil (Table 1). The soil cation-exchange capacity and organic matter content were also lower in Saemangeum soil compared to field soil whereas sodium content was higher in all samples of Saemangeum soil compared to field soil.
Production of AMF inoculum is influenced by various factors such as amount and type of fertilizer, host plant, climate and soil texture [29]. Among these, fertilizer is shown to be an important limiting factor of mycorrhizal colonization. Although there is no particular level after which phosphorous limits AMF infection to plants, high amount of phosphorous was shown to decrease the necessity for AMF and host plant symbiosis [30]. Although host plant is not as limiting in AMF sporulation, multiple host plants might increase the chances of high level of colonization [31]. In the present study, we have chosen low nutrient salt affected Saemangeum reclaimed land to isolate and propagate AMF spores. Soil analysis of this reclaimed land suggested that the low nutrient content of this soil is not favorable for the growth of most plant species. In addition, the unequal distribution of soil salinity in the area prohibits crop establishment and spore production. Thus, isolation and propagation of indigenous microorganisms might help to reintroduce native AMF inocula to improve plant growth in
3.2. Slide method of inoculation In the slide method of inoculation, the AMF spore germination and hyphae growth were closely monitored (Fig. 2). From among the 150 inoculants, six inoculants were able to germinate in vitro. Spore germination was visually monitored without disturbing the contents. The highest spore count was observed in Claroideoglomus lamellosum S-11 followed by Gigaspora margarita S-23, both of
Table 1 Soil properties of samples collected from Saemangeum reclaimed land containing healthy spores and field soil. Sample
pH
SS1 SS2 SS3 SS4 SS5 SS6 SS7 SS8 SS9 SS10 FS
7.91 7.44 6.85 6.58 6.49 6.54 6.25 6.20 6.32 6.36 6.30
EC
CEC
(dS/m) ± ± ± ± ± ± ± ± ± ± ±
0.10 0.04 0.14 0.03 0.01 0.07 0.03 0.02 0.02 0.04 0.01
0.30 0.32 0.60 0.43 0.24 0.34 0.36 1.60 1.40 1.13 1.21
± ± ± ± ± ± ± ± ± ± ±
(cmol/kg) 0.02 0.06 0.03 0.03 0.02 0.02 0.05 0.22 0.04 0.08 0.06
2.92 2.94 3.02 2.84 3.14 3.13 2.70 2.31 2.57 3.18 5.57
± ± ± ± ± ± ± ± ± ± ±
0.07 0.01 0.02 0.02 0.05 0.03 0.04 0.04 0.10 0.08 0.04
Organic matter
T-N
g/kg
(%)
3.88 4.26 5.28 3.88 2.82 5.08 5.18 4.03 3.66 3.54 23.62
± ± ± ± ± ± ± ± ± ± ±
0.09 0.30 0.06 0.05 0.08 0.39 0.41 0.35 0.26 0.14 0.78
0.026 0.026 0.033 0.026 0.025 0.032 0.027 0.023 0.012 0.018 0.142
± ± ± ± ± ± ± ± ± ± ±
0.003 0.001 0.002 0.001 0.002 0.001 0.001 0.001 0.001 0.000 0.001
P2O5 content
Kþ
(mg/kg)
(cmol/kg)
34.24 20.69 27.11 32.82 19.98 53.50 32.10 24.97 19.26 27.11 642.77
± ± ± ± ± ± ± ± ± ± ±
5.66 3.11 2.85 1.89 1.89 2.47 3.27 0.71 1.24 2.57 25.75
0.31 0.28 0.32 0.33 0.37 0.26 0.27 0.29 0.36 0.40 0.26
Ca2þ
± ± ± ± ± ± ± ± ± ± ±
0.02 0.00 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.00
0.47 0.62 0.54 0.45 0.44 1.40 0.71 0.67 0.26 0.43 4.11
Naþ
Mg2þ
± ± ± ± ± ± ± ± ± ± ±
0.05 0.01 0.02 0.02 0.02 0.04 0.03 0.00 0.01 0.01 0.06
1.04 0.95 1.18 1.04 1.09 0.75 1.15 0.75 0.63 1.07 0.60
± ± ± ± ± ± ± ± ± ± ±
0.08 0.01 0.04 0.04 0.03 0.02 0.05 0.01 0.01 0.03 0.01
0.39 0.51 0.64 0.80 0.74 0.19 0.31 0.78 1.41 1.26 0.21
± ± ± ± ± ± ± ± ± ± ±
0.03 0.00 0.02 0.03 0.02 0.01 0.01 0.01 0.03 0.04 0.00
SS e Saemangeum soil, FS e Field soil. Each value represents the mean of 3 replications ± standard error (SE). EC e electrical conductivity, CEC e cation exchange capacity, T-N e total nitrogen, P2O5 e phosphorus pentoxide, Kþ e potassium, Ca2þ e calcium, Mg2þ e magnesium, Naþ e sodium.
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Fig. 2. AMF spore germination and hyphae growth in slide method. (a) (b) and (c) spore germination. S e spore, H e hypha, I e infection site.
Table 2 Spore count, mycorrhizal colonization and arbuscule abundance in propagation pots. Inoculum
Spore count (100 g soil)
Colonization (M%)
Gigaspora margarita S-9 Claroideoglomus lamellosum S-11 Gigaspora margarita S-17 Gigaspora margarita S-20 Gigaspora margarita S-21 Gigaspora margarita S-23 Control
28.67 ± 5.46 391.33 ± 12.24 75.33 ± 8.51 98.67 ± 2.91 72.00 ± 0.67 235.33 ± 6.36 0
82.83 33.87 30.83 63.33 33.50 73.17 0
± ± ± ± ± ±
3.49 1.64 3.47 25.43 1.04 1.33
Arbuscules (A%) 56.23 12.07 12.33 75.00 11.12 48.63 0
± ± ± ± ± ±
3.45 1.79 0.37 1.75 1.21 3.38
Each value represents the mean of 3 replications ± standard error (SE).
Fig. 3. Phylogenetic relationships of AMF propagated in slide method based on 18S rDNA sequencing and related nearest neighbor sequences. The tree was constructed using closely related sequences based on Euclidean distance and Bootstrap values higher than 50% are shown. Spores from Saemangeum reclaimed soil are shown in bold. Number in parenthesis are the nucleotide accession numbers in Genbank.
this reclaimed land. Production of AMF inocula have been achieved through several methods which includes in vitro hairy root method [14], in vivo substrate based [32] and hydrophonic [18] culture methods. The substrate-based method is the most widely used method as it provides closer to field condition. Panwar et al. [22] developed a new technique for AMF mass production and reported that the spore developmental stages were visually monitored in vitro without disturbing the internal contents. We have also visually
monitored spore germination and hyphal elongation in gnotobiotic conditions. The slide method enabled us to understand the spore germination pattern and colonization of different species of AMF without disturbing the setup. Aseptic growth condition of this technique ensures the exclusion of any unwanted interaction with other microorganisms. This single spore culture technique could be useful for studying the preinfection stages of AMF as the plant roots could be examined frequently under low magnification. In addition, single spore cultures of AMF are a valuable resource for taxonomic
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and biochemical studies of new AMF species as they produce more homogenous spores of certain species. Inocula of AMF production from single spore pot culture method was initially employed by Brundrett and Juniper [33] who reported an open-filter technique for assessment of spore germination using cellulose-acetate filter pieces. An effective inoculum contains mycorrhizal spores, colonized root bits and hyphae. Our study demonstrated that slide method resulted in AMF propagation up to 391 spores per 100 g soil. Using Clay-brick granules and river sand as substrate Gaur and Adholeya [34] produced up to 880 infective propagules of Glomus intraradices per 100 ml of substrate. Schlemper and Sturmer [35] recently reported that up to 240 spores per 100 cm3 soil (lignocellulosic agrowastes mixed with sand:rice shell) were produced. Through an on-farm production technique Douds et al. [11] reported that they were able to obtain up to 2150 propagule cm3. Among these various methods, the onfarm production method seem to be the most effective in terms of high number of propagules production. The use of indigenous species and continuous crop rotation might have triggered the activity of AMF in the on-farm production method. On the other hand, the pure cultures produced in the slide method can be used for further morphological and molecular studies. In addition, pure cultures enable one to understand the effect of individual AMF species on plant growth. We found that in some pots the number of spores were less after successive propagation cycles. Continuous use of the same host plant might have limited the mycorrhizal symbiosis, however, altering the host plants either from C3 to C4 or vice versa may help to overcome this problem. Our results demonstrated that production of AMF species pure culture can be achieved using single spores as inocula through the slide method. 5. Conclusion Slide method was convenient to visually monitor the spore germination and establishment. Our results demonstrated that production of AMF species in pure culture can be achieved using single spore as inoculum through the slide method and is highly reproducible. Further studies on the molecular aspects of factors which most likely stimulate spore production will be critical on propagating this obligate biotroph. The pure cultures of indigenous AMF isolates can be used as potential plant growth promoting fungal inocula. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2015R1A2A1A05001885). References [1] Y. Shachar-Hill, P.E. Pfeffer, D.D. Douds, S.F. Osman, L.W. Doner, R.G. Ratcliffe, Partitioning of intermediary carbon metabolism in vesicular-arbuscular mycorrhizal leek, Plant Physiol. 108 (1995) 7e15. [2] S.E. Smith, D.J. Read, Mycorrhizal Symbiosis, second ed., Academic Press, San Diego, CA, 1997. [3] M.P. Sharma, A. Adholeya, Enhanced growth and productivity following inoculation with indigenous AM fungi in various varieties of onion (Allium cepa L.) in an alfisol, Biol. Agric. Hortic. 18 (2000) 1e14. [4] X.L. Sui, A.R. Li, Y. Chen, K.Y. Guan, L. Zhuo, Y.Y. Liu, Arbuscular mycorrhizal fungi: potential biocontrol agents against the damaging root hemiparasite Pedicularis kansuensis? Mycorrhiza 24 (2014) 187e195.
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22 (2003) 283e287. [33] M. Brundrett, S. Juniper, Non-destructive assessment of spore germination of VAM fungi and production of pot cultures from single spores, Soil Biol. Biochem. 27 (1995) 85e91. [34] A. Gaur, A. Adholeya, Effects of the particle size of soil-less substrates upon
AM fungus inoculum production, Mycorrhiza 10 (2000) 43e48. [35] T.R. Schlemper, S.L. Sturmer, On farm production of arbuscular mycorrhizal fungi inoculum using lignocellulosic agrowastes, Mycorrhiza 24 (2014) 571e580.
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Propagation technique of arbuscular mycorrhizal fungi isolated from coastal reclamation land Gopal Selvakumar, Ramasamy Krishnamoorthy 1, Kiyoon Kim, Tongmin Sa* Department of Environmental and Biological Chemistry, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 November 2015 Received in revised form 2 March 2016 Accepted 14 March 2016
Arbuscular mycorrhizal fungi (AMF) are well known for their plant growth promoting potential and stress tolerance ability. AMF promotes plant growth even at adverse environmental conditions and improve plant nutrient uptake. This study aimed to propagate AMF using a single spore inoculation technique. Soil samples were collected from salt affected Saemangeum reclaimed land. Collected soil samples were subjected to soil analysis. In the single spore inoculation method, sorghum sudangrass (Sorghum bicolor L.) was inoculated with a single spore. Among the 150 inoculants, six spores were able to germinate in vitro. Geminated spores were transferred to 1 kg pots containing sterilized field soil. After 120 days, the contents were mixed and transferred to 2.5 kg pots and maintained for another 120 days. After 240 days, spore count and colonization were checked. The propagated spores were identified using nested PCR followed by sequencing. The 18S rDNA sequencing of spores revealed that the spores belonged to Gigaspora margarita and Claroideoglomus lamellosum. Among the 6 inoculants, Claroideoglomus lamellosum S-11 (391 spore per 100 g of soil) had the highest spore count followed by Gigaspora margarita S-23 (235 spore per 100 g of soil). Slide method allowed visual monitoring of spore germination in vitro as well as being able to mass produce pure cultures of AMF for bio-inoculation purposes. © 2016 Elsevier Masson SAS. All rights reserved.
Handling Editor: Prof. C.C. Tebbe Keywords: Mass production Arbuscular mycorrhizal fungi Slide method Gigaspora margarita Claroideoglomus lamellosum
1. Introduction Arbuscular mycorrhizal fungi (AMF) are widespread soil fungi which form mutualistic symbiosis with more than 80% of land plants and are believed to be obligate symbiotic biotrophs. Only the intraradical hypha of this fungus takes up hexose as carbon source in the apoplast of the plant root cortex cells to complete their life cycle and sporulate [1]. Therefore, the ability of AMF to utilize externally supplied carbon source in asymbiotic medium is limited. Although AMF are indigenous to agricultural soils, inoculation of these fungi improves plant growth by enhancing the uptake of immobile soil mineral nutrients and water [2]. AMF have increased the growth of various field-grown crops, including cotton, tomato, orange, pepper and onions [3]. In addition to enhancement of
* Corresponding author. Department of Environmental and Biological Chemistry, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea. E-mail address: [email protected] (T. Sa). 1 Present address: Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai, India. http://dx.doi.org/10.1016/j.ejsobi.2016.03.005 1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
nutrient uptake by plants, AMF also help plants withstand various biotic and abiotic stresses such as pathogen attack [4], drought stress [5] and salt stress [6]. Enhanced plant growth through AMF inoculation can be ensured by two principal ways (1) selection and inoculation of efficient mycorrhizal fungi and (2) promotion of the efficient native mycorrhizal fungi [7]. Although previous reports support that some AMF are beneficial to a wide variety of plant species [8], some reports argue that host specificity of AMF might reduce the mycorrhizal interaction between plant and fungi [9,10]. AMF inocula that are commercially available come in a variety of forms at low to high concentration and from single inoculum to multiple combined inoculum in a carrier material. However, their performance in nonindigenous soil tends to decrease due to their lack of host specificity and differences in agricultural management practices [11]. Estrada et al. [12] reported that native AMF species from Mediterranean saline soil are more effective in improving plant growth than the introduced AMF species. In addition, reintroduction of native AMF inocula may be necessary to overcome the harmful effects of previous agricultural management, e.g. excessive use of fungicide. Purchasing large amounts of inoculum necessary for large-scale
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agriculture is not cost-effective. Moreover, even though inocula of AMF are commercially available, production of pure cultures of host specific AMF is essential. Development of pure culture also enables the study of the morphological characteristics of new AMF species and their individual effect on plant growth. Previously, pure cultures of AMF have been obtained in vitro using Ri-TDNA transformed root organ culture [13,14], aeroponics [15,16] and bicompartmental Petri plates [17]. However, these in vitro methods limit their application in the field due to their requirement of precolonized plants and different growth medium [18]. Although pot culture and monoxenic culture propagated spores share similar ontogeny, daughter spores from monoxenic cultures usually differentiate into smaller spores with paler pigmentation and thinner laminated spore walls [19,20]. The inocula produced by substrate-free cultivation method may require carrier material before their application in large scale to improve their survivability. In addition, inocula produced through soil-based method are least artificial, most adopted and can be applied directly in the field [11]. The present study aimed to propagate the AMF spores isolated from Saemangeum reclaimed land using single spores as starter inoculum. 2. Materials and methods 2.1. Study area and soil sample collection Saemangeum is one of the world's largest reclamation sites adding about 400 km2 to South Korea's total geographical area. A total of thirty five rhizosphere soil samples (10 cm radius and 15 cm depth; approximately, one kg for each sample) have been collected from Saemangeum reclamation land. The distance between each sample was at least 10 m, so that the chances of sharing the same AMF species between samples are reduced. Based on the initial spore count, ten soil samples which had over 10 healthy spores were used in this study. Soils from rhizospheres of dominant plant species such as Phragmites australis, Cyperus polystachyos and Miscanthus sinensis were chosen to obtain different AMF isolates. Each rhizosphere soil sample along with the plant roots was collected in a sterilized polyvinyl chloride bag and transported immediately to the laboratory in icebox and kept at 4 C until use. 2.2. Soil analysis and initial spore enumeration All the collected soil samples were subjected to initial spore count and determination of electrical conductivity (EC) value (1:5 dilution method). The initial spore count of the soil samples were assessed using wet sieving and decanting method as described by Daniels and Skipper [21] followed by sucrose centrifugation method. Soil properties such as pH, cation exchange capacity (CEC), organic matter, micro and macro nutrient contents were examined. 2.3. Single spore - slide method Freshly isolated spores were examined under the microscope and only the healthy and damage-free spores were used for propagating the AMF in vitro. Slide method was conducted to visually monitor spore germination. Briefly, sterilized filter paper (Whatman No. 6) was excised to ¾ of the glass slide and tied with rubber band and kept in a 50 ml sterile falcon tube containing the mixture of plant root exudates and sterilized Hoagland's nutrient solution. The bottom of the filter paper was in contact with the solution so that the moisture can be maintained throughout the experiment. The plant root exudates were obtained as described in Panwar et al. [22]. Sorghum sudangrass seeds were surface sterilized by immersing them in 70% ethanol for 2 min followed by 1% sodium
hypochlorite (NaOCl) for 3 min and thoroughly rinsed with sterile distilled water for seven to ten times. The surface sterilized seeds were placed in a petri dish containing sterile moist filter paper and kept in the growth chamber for 2e3 days. The growth chamber conditions were as follows: 12 h of light at 25 C and 12 h of dark at 20 C and maintenance of 70% humidity. The germinated seeds were transferred to slides and tied with rubber band without harming the radicles. A healthy single spore was placed near the growing root (Fig. 1). A total of 150 inoculants were made. The setup was kept in the growth chamber with the above mentioned conditions for two weeks. Every 3 days the spore was examined under the microscope for germination. After 2 weeks, six slides with successfully germinated spores were transferred to 1 kg pots containing sterilized soil without disturbing the setup and new pregerminated sorghum seeds were sown. Sterilized field soil consecutively autoclaved for three days was added to fill up the remaining space in the pots. New pre-germinated sorghum seeds were sown and the plants were allowed to grow for 120 days. Hoagland's nutrient solution was poured on every pot every week. After 105 days, the plants were subjected to drought stress by withholding water or Hoagland's nutrient solution for 15 days to induce spore production. After 120 days, the soil from each pot was mixed and transferred to 2.5 kg pot with sterilized soil. Pre-germinated sorghum seeds were sown and grown for another 120 days following the above mentioned procedure. After 240 days, root and soil samples were collected and examined for mycorrhizal colonization and spore numbers. Briefly, the roots were first washed with 10% KOH for 10 min in water bath at 90 C. Then the roots were washed with tap water and immersed in 2% HCl for 10 min at room temperature. After discarding the HCl, 0.5% trypan blue in lactoglycerol was added and the roots were allowed to stain at 90 C for 10 min. The staining solution was discarded and the stained roots were washed with tap water. Roots were immersed in destaining solution for overnight to remove excess staining and were checked for colonization [23]. The stained root fragments (1 cm) were arranged in glass slides and observed under the microscope for the presence of hypha, vesicles and arbuscules. Scoring was done based on the intensity of colonization (0e5) and based on the arbuscules intensity (A0eA3) as described by Trouvelot et al. [24]. A total of 30 root fragments were observed for each treatment. Intensity of the mycorrhizal root colonization was estimated as the amount of cortex cell that colonized by mycorrhiza relative to the whole root system (M%). Abundance of arbuscule was estimated as the arbuscule richness in the whole root system (A%). The Mycocalc software was used to determine the M%, and A%. 2.4. 18S rDNA sequencing Five healthy spores from successfully propagated slide method pots were taken in microcentrifuge tube and surface sterilized with 2% chloramine-T and 100 mg/ml streptomycin for 30 min. Then the spores were transferred to a sterilized PCR tube containing 10 ml of 1:1 ratio of 10X PCR buffer and sterilized distilled water. Spores were aseptically crushed with a sterilized blunt end pasteur pipette. The 18S rDNA of arbuscular mycorrhizal fungal spores were amplified using the nested PCR [25]. In the first round of PCR, eukaryotic genes were targeted using universal eukaryotic primers GeoA2 (50 -CCAGTAGTCATATGCTTGTCTC-30 ) and Geo11 (50 ACCTTGTTACGACTTTTACTTCC-30 ) which amplified the genes with the product length of 1800 bp. The final product was diluted 1:100 ratio with TE buffer [26] and used as template for next PCR. In the second round of PCR, AMF specific primer AM1 (50 GTTTCCCGTAAGGCGCCGAA-30 ) was used to amplify AMF specific
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41
Fig. 1. Single spore inoculation, slide method. (a) aseptically germinated seedling tied on a glass slide covered with sterilized filter paper secured, (b) slide kept in falcon tube containing root exudates, (c) freshly isolated AMF spore inoculated near growing root of sorghum Sudangrass. SS e Sorghum-sudan grass seedling, RE e root exudate, R e root and S e spore.
region along with NS31 (50 -TTGGAGGGCAAGTCTGGTGCC-30 ) targeting the product size of 550 bp. Nearly complete 18S rDNA sequences were aligned and the closest fungal identities were obtained after deduced by BLAST search. The phylogenetic tree was generated after multiple alignment with CLUSTAL-W [27] and the tree topologies were evaluated by bootstrap analysis of 1000 dataset using MEGA version 6.0 software [28]. The nucleotide sequence of 18S rDNA sequences were deposited in GenBank and the accession numbers were given as KP677594 to KP677599.
these inoculants propagated over 200 spores per 100 g of soil (Table 2). However, the highest mycorrhizal colonization was observed in Gigaspora margarita S-9 followed by Gigaspora margarita S-23. The highest arbuscule abundance was observed in Gigaspora margarita S-20 followed by Gigaspora margarita S-9. 3.3. 18S rDNA sequencing Six inoculants were taken for further identification by amplifying the 18S region of fungal DNA and their closest neighbors were identified (Fig. 3). Five of the isolates belong to Gigaspora margarita and one isolate belong to Claroideoglomus lamellosum.
3. Results 3.1. Soil analysis and initial spore enumeration
4. Discussion
The EC value of the Saemangeum soil samples varied from 0.30 to 1.60 dS/m. Most of the spores were observed to be damaged probably due to the salinity level of Saemangeum soil. The soil physico-chemical properties show that nitrogen, phosphorous and calcium contents of Saemangeum reclaimed soil is very low compared to normal field soil (Table 1). The soil cation-exchange capacity and organic matter content were also lower in Saemangeum soil compared to field soil whereas sodium content was higher in all samples of Saemangeum soil compared to field soil.
Production of AMF inoculum is influenced by various factors such as amount and type of fertilizer, host plant, climate and soil texture [29]. Among these, fertilizer is shown to be an important limiting factor of mycorrhizal colonization. Although there is no particular level after which phosphorous limits AMF infection to plants, high amount of phosphorous was shown to decrease the necessity for AMF and host plant symbiosis [30]. Although host plant is not as limiting in AMF sporulation, multiple host plants might increase the chances of high level of colonization [31]. In the present study, we have chosen low nutrient salt affected Saemangeum reclaimed land to isolate and propagate AMF spores. Soil analysis of this reclaimed land suggested that the low nutrient content of this soil is not favorable for the growth of most plant species. In addition, the unequal distribution of soil salinity in the area prohibits crop establishment and spore production. Thus, isolation and propagation of indigenous microorganisms might help to reintroduce native AMF inocula to improve plant growth in
3.2. Slide method of inoculation In the slide method of inoculation, the AMF spore germination and hyphae growth were closely monitored (Fig. 2). From among the 150 inoculants, six inoculants were able to germinate in vitro. Spore germination was visually monitored without disturbing the contents. The highest spore count was observed in Claroideoglomus lamellosum S-11 followed by Gigaspora margarita S-23, both of
Table 1 Soil properties of samples collected from Saemangeum reclaimed land containing healthy spores and field soil. Sample
pH
SS1 SS2 SS3 SS4 SS5 SS6 SS7 SS8 SS9 SS10 FS
7.91 7.44 6.85 6.58 6.49 6.54 6.25 6.20 6.32 6.36 6.30
EC
CEC
(dS/m) ± ± ± ± ± ± ± ± ± ± ±
0.10 0.04 0.14 0.03 0.01 0.07 0.03 0.02 0.02 0.04 0.01
0.30 0.32 0.60 0.43 0.24 0.34 0.36 1.60 1.40 1.13 1.21
± ± ± ± ± ± ± ± ± ± ±
(cmol/kg) 0.02 0.06 0.03 0.03 0.02 0.02 0.05 0.22 0.04 0.08 0.06
2.92 2.94 3.02 2.84 3.14 3.13 2.70 2.31 2.57 3.18 5.57
± ± ± ± ± ± ± ± ± ± ±
0.07 0.01 0.02 0.02 0.05 0.03 0.04 0.04 0.10 0.08 0.04
Organic matter
T-N
g/kg
(%)
3.88 4.26 5.28 3.88 2.82 5.08 5.18 4.03 3.66 3.54 23.62
± ± ± ± ± ± ± ± ± ± ±
0.09 0.30 0.06 0.05 0.08 0.39 0.41 0.35 0.26 0.14 0.78
0.026 0.026 0.033 0.026 0.025 0.032 0.027 0.023 0.012 0.018 0.142
± ± ± ± ± ± ± ± ± ± ±
0.003 0.001 0.002 0.001 0.002 0.001 0.001 0.001 0.001 0.000 0.001
P2O5 content
Kþ
(mg/kg)
(cmol/kg)
34.24 20.69 27.11 32.82 19.98 53.50 32.10 24.97 19.26 27.11 642.77
± ± ± ± ± ± ± ± ± ± ±
5.66 3.11 2.85 1.89 1.89 2.47 3.27 0.71 1.24 2.57 25.75
0.31 0.28 0.32 0.33 0.37 0.26 0.27 0.29 0.36 0.40 0.26
Ca2þ
± ± ± ± ± ± ± ± ± ± ±
0.02 0.00 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.00
0.47 0.62 0.54 0.45 0.44 1.40 0.71 0.67 0.26 0.43 4.11
Naþ
Mg2þ
± ± ± ± ± ± ± ± ± ± ±
0.05 0.01 0.02 0.02 0.02 0.04 0.03 0.00 0.01 0.01 0.06
1.04 0.95 1.18 1.04 1.09 0.75 1.15 0.75 0.63 1.07 0.60
± ± ± ± ± ± ± ± ± ± ±
0.08 0.01 0.04 0.04 0.03 0.02 0.05 0.01 0.01 0.03 0.01
0.39 0.51 0.64 0.80 0.74 0.19 0.31 0.78 1.41 1.26 0.21
± ± ± ± ± ± ± ± ± ± ±
0.03 0.00 0.02 0.03 0.02 0.01 0.01 0.01 0.03 0.04 0.00
SS e Saemangeum soil, FS e Field soil. Each value represents the mean of 3 replications ± standard error (SE). EC e electrical conductivity, CEC e cation exchange capacity, T-N e total nitrogen, P2O5 e phosphorus pentoxide, Kþ e potassium, Ca2þ e calcium, Mg2þ e magnesium, Naþ e sodium.
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Fig. 2. AMF spore germination and hyphae growth in slide method. (a) (b) and (c) spore germination. S e spore, H e hypha, I e infection site.
Table 2 Spore count, mycorrhizal colonization and arbuscule abundance in propagation pots. Inoculum
Spore count (100 g soil)
Colonization (M%)
Gigaspora margarita S-9 Claroideoglomus lamellosum S-11 Gigaspora margarita S-17 Gigaspora margarita S-20 Gigaspora margarita S-21 Gigaspora margarita S-23 Control
28.67 ± 5.46 391.33 ± 12.24 75.33 ± 8.51 98.67 ± 2.91 72.00 ± 0.67 235.33 ± 6.36 0
82.83 33.87 30.83 63.33 33.50 73.17 0
± ± ± ± ± ±
3.49 1.64 3.47 25.43 1.04 1.33
Arbuscules (A%) 56.23 12.07 12.33 75.00 11.12 48.63 0
± ± ± ± ± ±
3.45 1.79 0.37 1.75 1.21 3.38
Each value represents the mean of 3 replications ± standard error (SE).
Fig. 3. Phylogenetic relationships of AMF propagated in slide method based on 18S rDNA sequencing and related nearest neighbor sequences. The tree was constructed using closely related sequences based on Euclidean distance and Bootstrap values higher than 50% are shown. Spores from Saemangeum reclaimed soil are shown in bold. Number in parenthesis are the nucleotide accession numbers in Genbank.
this reclaimed land. Production of AMF inocula have been achieved through several methods which includes in vitro hairy root method [14], in vivo substrate based [32] and hydrophonic [18] culture methods. The substrate-based method is the most widely used method as it provides closer to field condition. Panwar et al. [22] developed a new technique for AMF mass production and reported that the spore developmental stages were visually monitored in vitro without disturbing the internal contents. We have also visually
monitored spore germination and hyphal elongation in gnotobiotic conditions. The slide method enabled us to understand the spore germination pattern and colonization of different species of AMF without disturbing the setup. Aseptic growth condition of this technique ensures the exclusion of any unwanted interaction with other microorganisms. This single spore culture technique could be useful for studying the preinfection stages of AMF as the plant roots could be examined frequently under low magnification. In addition, single spore cultures of AMF are a valuable resource for taxonomic
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and biochemical studies of new AMF species as they produce more homogenous spores of certain species. Inocula of AMF production from single spore pot culture method was initially employed by Brundrett and Juniper [33] who reported an open-filter technique for assessment of spore germination using cellulose-acetate filter pieces. An effective inoculum contains mycorrhizal spores, colonized root bits and hyphae. Our study demonstrated that slide method resulted in AMF propagation up to 391 spores per 100 g soil. Using Clay-brick granules and river sand as substrate Gaur and Adholeya [34] produced up to 880 infective propagules of Glomus intraradices per 100 ml of substrate. Schlemper and Sturmer [35] recently reported that up to 240 spores per 100 cm3 soil (lignocellulosic agrowastes mixed with sand:rice shell) were produced. Through an on-farm production technique Douds et al. [11] reported that they were able to obtain up to 2150 propagule cm3. Among these various methods, the onfarm production method seem to be the most effective in terms of high number of propagules production. The use of indigenous species and continuous crop rotation might have triggered the activity of AMF in the on-farm production method. On the other hand, the pure cultures produced in the slide method can be used for further morphological and molecular studies. In addition, pure cultures enable one to understand the effect of individual AMF species on plant growth. We found that in some pots the number of spores were less after successive propagation cycles. Continuous use of the same host plant might have limited the mycorrhizal symbiosis, however, altering the host plants either from C3 to C4 or vice versa may help to overcome this problem. Our results demonstrated that production of AMF species pure culture can be achieved using single spores as inocula through the slide method. 5. Conclusion Slide method was convenient to visually monitor the spore germination and establishment. Our results demonstrated that production of AMF species in pure culture can be achieved using single spore as inoculum through the slide method and is highly reproducible. Further studies on the molecular aspects of factors which most likely stimulate spore production will be critical on propagating this obligate biotroph. The pure cultures of indigenous AMF isolates can be used as potential plant growth promoting fungal inocula. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2015R1A2A1A05001885). References [1] Y. Shachar-Hill, P.E. Pfeffer, D.D. Douds, S.F. Osman, L.W. Doner, R.G. Ratcliffe, Partitioning of intermediary carbon metabolism in vesicular-arbuscular mycorrhizal leek, Plant Physiol. 108 (1995) 7e15. [2] S.E. Smith, D.J. Read, Mycorrhizal Symbiosis, second ed., Academic Press, San Diego, CA, 1997. [3] M.P. Sharma, A. Adholeya, Enhanced growth and productivity following inoculation with indigenous AM fungi in various varieties of onion (Allium cepa L.) in an alfisol, Biol. Agric. Hortic. 18 (2000) 1e14. [4] X.L. Sui, A.R. Li, Y. Chen, K.Y. Guan, L. Zhuo, Y.Y. Liu, Arbuscular mycorrhizal fungi: potential biocontrol agents against the damaging root hemiparasite Pedicularis kansuensis? Mycorrhiza 24 (2014) 187e195.
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