African Journal of
Agricultural Research

  • Abbreviation: Afr. J. Agric. Res.
  • Language: English
  • ISSN: 1991-637X
  • DOI: 10.5897/AJAR
  • Start Year: 2006
  • Published Articles: 6362

Full Length Research Paper

Effect of arbuscular mycorrhizal fungi on survival and growth of micropropagated Comanthera mucugensis spp. mucugensis (Eriocaulaceae)

Lidiane Silva Pereira
  • Lidiane Silva Pereira
  • Department of Biological Sciences, State University of Santa Cruz, 45662-900, Ilhéus, Bahia, Brazil
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Irailde da Silva Santos
  • Irailde da Silva Santos
  • Department of Biological Sciences, State University of Feira de Santana, UEFS, Feira de Santana, 44036-900 Ilhéus, Bahia, Brazil
  • Google Scholar
Fátima Cerqueira Alvim
  • Fátima Cerqueira Alvim
  • Department of Agricultural and Environmental Sciences, State University of Santa Cruz, 45662-900, Ilhéus, Bahia, Brazil
  • Google Scholar
José Olimpio de Souza Júnior
  • José Olimpio de Souza Júnior
  • Department of Agricultural and Environmental Sciences, State University of Santa Cruz, 45662-900, Ilhéus, Bahia, Brazil
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Eduardo Gross
  • Eduardo Gross
  • Department of Agricultural and Environmental Sciences, State University of Santa Cruz, 45662-900, Ilhéus, Bahia, Brazil
  • Google Scholar


  •  Received: 09 November 2016
  •  Accepted: 19 January 2017
  •  Published: 18 May 2017

 ABSTRACT

The use of micropropagation technique has been an alternative to conservation of endangered species, Comanthera mucugensis subsp. mucugensis (popular namely sempre viva de Mucuge); however, there is no information on the effect of arbuscular mycorrhizal fungi (AMF) on the acclimation process of micropropagated plants. This study evaluated the survival, growth and nutritional aspects of the species, C. mucugensis subsp. mucugensis inoculated with native AMFs in greenhouse condition. The design of the experiment consisted initially of 80 sampling units divided into four treatments: plants inoculated with native AMF, with microbiota filtrate from soil, with AMF plus filtrate and control (non-inoculated plants). At three and eleven-month-old, the plants were collected for evaluation of growth, nutrition and mycorrhizal colonization. After eleven months of experiment, survival rate of AMF and AMF plus filtrate plants were 62.5 and 87.5%, respectively, and only one microbiota filtrate and one control plants survived. AMF inoculation also provided increase in n dry matter of rosettes and permitted obtaining flowering ten-month-growth plants. Rates of mycorrhizal colonization were high at three (aproximately 64.9%) and eleven (aproximately 94.5%) months for AMF and AMF plus filtrate plants. Number of spores in rhizosphere soil of mycorrhizal plants was also high (1599 per 100 dm3 of soil) and seven diferent species of AMF were identified at the end of experiment. Data set evidenced mycortrophic character of C. mucugensis subsp. mucugensis and the importance of AMF inoculation for acclimation and survival of microprogagated plants which is essential for conservation of this endangered plant.

 

Key words: Micropropagation, nutrition, arbuscular mycorrhiza fungi, sempre viva de Mucuge, aclimatation 


 INTRODUCTION

The Eriocaulaceae family comprises eleven genera and ca. 1200 species, has a pantropical distribution (Echternacht  et   al.,   2010),  and  presents  its  diversity center on the Espinhaço Range between Minas Gerais and Bahia (Giulietti and Hensold, 1990; Sano, 2004). About 70% of the total Brazilian species of Eriocaulaceae occur at Espinhaço Range, 85% are endemic and often are restricted to a single mountain (Giulietti et al., 2005; Costa et al., 2008). The species, Comanthera mucugensis subsp. mucugensis is one of this microendemic Eriocaulaceae plants that occured on municipality of Mucuge (Bahia) at eastern side of the Chapada Diamantina region. This species is popularly known as sempre viva de Mucuge (evergreen of Mucuge) and its inflorescence remains with the same color and shape when their scapes, chapters and flowers are collected for making dried floral arrangements. At region of rupestrian field on Mucuge where these plants occur naturally, they were one of the main sources of income for local inhabitants at the mid-twentieth century, and each year were sold tons of flowers, especially to Europe and the United States (MMA/PNMA, 1996), whichreduced the natural population since the flowers are still at anthesis when collected to be sold as ornamental (Giulietti et al., 1988; Cerqueira et al., 2008).
 
Recently, C. mucugensis subsp. mucugensis was prohibited from being collected because their exploitation has been carried out without planning and without any control or cultivation (Lima-Brito et al., 2016), and currently, this plant is on the Official List of Species of the Brazilian Flora Endangered (MMA, 2008). Some tentatives of plant management are already being developed at Parque Municipal de Mucuge, as to protect C. mucugensis subsp. mucugensis populations and promote the propagation and cultivation, seeking alternative sources of income to the population of the municipality (Paixão-Santos et al., 2003; Ramos et al., 2005; Teixeira and Linsker 2005).
 
With the aim to increase C. mucugensis subsp. mucugensis populations in Mucuge region, the micropropagation technique has been used as a viable option for the production of seedlings of this species (Lima-Brito et al., 2011; Pêgo et al., 2013). Despite the advantages in using this technique, there are still some obstacles to their wide application, especially as regards acclimation, that is, the conditions to be transplanted in vitro to greenhouse, since mortality rate of C. mucugensis subsp. mucugensis micropropagated plants is high.
 
The absence of beneficial soil microorganisms can result to negative effects on the plant acclimation process due low adaptation to new environmental conditions imposed (Borkowska, 2002). Studies on the association of arbuscular mycorrhizal fungi (AMF) with some agronomic and ornamental plants demonstrate benefits of these microorganisms as plant growth regulators and their importance to management and acclimation (Rocha et al., 2006; Yadav et al., 2013; Moreira et al., 2015; Villarreal   et   al.,  2016).   Arbuscular   mycorrhizal  fungi (AMF) are an important microbial group of the soil, which form a mutualistic symbiosis with the roots of plants affecting several processes and functions in the ecosystem such as nutrient cycling, plant productivity and competition (Hazard et al., 2013). This microorganism have been used as an alternative to increase the resilience of many species during the acclimation process, stimulating the autotrophic stage of transition from in vitro to soil and influencing morphogenesis and architecture of root, ensuring a health formation and development of root system after transplanting (Zemke et al., 2003; Kapoor et al., 2008; Stancato and Silveira, 2010). Apart from this, AMF can act as biological controller of some pathogens and to reduce tensions as nutrition, availability of water and salinity involved on micropropagation (Schubert et al., 1990; Jaizme-Vega and Azcón, 1991).
 
In the present study, the authors evaluated native AMF and microbiota inoculation on acclimation of C. mucugensis subsp. mucugensis micropropagated plants, analysing survival and nutritional status with goal to contribute to process of population restoration of this endangered plant.


 MATERIALS AND METHODS

In vitro culture
 
In the experiment, 120 days old micropropagated plants of the C. mucugensis subsp. mucugensis obtained from the Vegetable Tissue Culture Laboratory of the Horto Florestal Experimental Unit, belonging to the Biological Sciences Department of the Feira de Santana State University, in the municipality of Feira of Santana, Bahia were used. The chemical characterization of the in vitro plants was carried out at the Laboratory of Analysis of Vegetable Tissues of the Cocoa Research Center (CEPEC) of the Executive Committee for Cocoa Plantation Planning (CEPLAC). The results were: N = 42.18 g.Kg−1; P = 1.98 g.Kg−1; K = 22.92 g.Kg−1; Ca = 2.29 g.Kg−1; Mg = 1.28 g.Kg−1; Cu = 2.33 mg.Kg−1; Fe = 38.12 mg.Kg−1; Mn = 44.3 mg.Kg−1; Zn = 46.84 mg.Kg−1.
 
Obtaining plant material
 
The experiment was conducted in a greenhouse at the University of Santa Cruz (Ilheus, BA) under natural conditions of temperature and luminosity. Micropropagated seedlings of C. mucugensis (Giul.) L.R.Parra & Giul. subsp. mucugensis were provided by the Tissue Culture Laboratory of the State University of Feira de Santana (UEFS), and grown in plastic pots containing 0.4 dm3 of soil collected at rupestrian field on Parque Municipal de Mucugê (Mucugê, Bahia, Brazil; 12°59’27’’S, 41°20’11’’W and 980 a.s.l). This native soil was previously sterilized at 121°C for two cycles of 1 h with 48 h interval, and after reaching ambient temperature, the resulting pH (measured in water) was 2.8 and it was not adjusted. Previous experiments liming on soil  and  substrate (coarse and fine sand) indicated that this plant do not tolerate (die) soil pH reaching 5.
 
The soil of rupestrian field collected for the experiment presented a sandy texture class with 84.80% sand, 15.06% silt and 0.14% clay. The chemical characterization of the soil was performed by the Chemical Analysis Laboratory of the Department of Soil Science College of Agriculture "Luiz de Queiroz", University of São Paulo (USP-ESALQ) following Raij et al. (2001) method and presented the following results: pH, 2.7 (in CaCl2); organic matter, 76 g dm−3; P, 5 mg dm−3; S, 2 mg dm−3; K, 0.4 mmolc dm−3; Ca, 2 mmolc dm−3; Mg, 2 mmolc dm−3; Al, 19 mmolc dm−3; H + Al, 386 mmolc dm−3; Cu, 0.1 mg dm−3; Mn, 1.1 mg dm−3; Fe, 30 mg dm−3; Zn, 1.8 mg dm−3.
 
Ten plants of C. mucugensis subsp. mucugensis were collected on field (from natural population at Mucuge) and evaluated for nutrient composition aiming to prescribe a nutritional fertilization previously to perform the experiment. Dry matter of the rosettes (leaves) was chemical characterized (Raij et al., 2001) and results were: N, 10.78; P, 0.27; K, 1.53; Ca, 0.55; Mg, 1.40; S 0.76 (all maconutrients at g kg−1); B, 4.67; Cu, 0.80; Fe, 26.20; Mn, 8.10; Zn, 4.60 (all micronutrients at mg kg−1).
 
Experimental design
 
The experimental design was completely randomized and initially 80 sampling units divided among the control and three treatments: plants inoculated with native AMF, with microbiota filtrate from soil, with AMF plus microbiota filtrate. In 20 replicates from each treatment, 12 were collected at three month plant growth to investigate the initial mycorrhiza establishment at the acclimatization phase. The remaining eight plants were collected at 11 month of plant growth. The spores of native AMF used as inoculum were obtained from the multiplication pot using C. mucugensis subsp. mucugensis as host plant, since there was low sporulation on the previous attempt using a conventional host plant (Brachiaria decumbes).
 
Spores were isolated from 100 g of soil using the technique of wet sieving of Gerdemann and Nicolson (1963) and centrifugation in 50% sucrose using the technique of Jenkins (1964). To simulate the natural microbial composition of soil, a filtrate was prepared using a suspension of field soil with autoclaved distilled water (1:10 m/v), which was stirred for 24 h (Sudová and Vosátka, 2008). Subsequently, the material was passed through a glass funnel containing filter paper (Whatman no. 1) with the aid of a vacuum pump retaining the solid part and mycorrhizal propagules.
 
After transplantation from in vitro condition to the platic pots in a greenhouse, the micropropagated plants (85 days old), and according to the treatment, received 10 ml suspension containing: mycorrhizal inoculum with 470 spores, microbiota filtrate and mycorrhizal inoculum and filtrate.
 
Fertilization of plants in pots
 
Every week, the plants were irrigated at intervals of 48 h with 30 mL of ¼ ionic strength nutrient solution adapted from Hoagland and Arno (1950). The irrigation with distilled water of the same volume was interspersed with nutrient solution. The complete nutrient solution (in mg L-1) consisted of: N, 70.00; P, 5; K, 45.36; Ca, 50; Mg, 12.16; S, 64.00; Zn, 0.01; B, 0.11; Cu, 0.005; Fe, 0.25; Mn, 0.11; Mo, 0.002 as H3BO3; MnSO4; ZnSO4; CuSO4.5H2O; (NH4)6 Mo7O24.4H2O; Fe-EDTA; KH2PO4; (NH4)2SO4; K2SO4; Ca(NO3)2; MgSO4 salts.
 
Dry biomass and nutritional analysis
 
For the analysis of biomass, rosettes (leaves) were dried at  60°C in an oven with forced air circulation until constant weight. Dry matter was obtained and due to the small volume of plant material, only one sample (the sum of all replicates) per treatment was sent to the Laboratory of Mineral Nutrition of Plants USP - ESALQ for nutritional analysis. The methodologies used in this analysis were: P: colorimetry (ammonium metavanadate method), S: colorimetry (turbidimetric barium sulfate), K, Ca and Mg by atomic absorption spectrophotometry, Cu, Fe, Mn and Zn: absorption spectrophotometry atomic; sulfuric digestion for total N, B: colorimetry (Azomethine H method).
 
Assessment of AMFs colonization
 
To estimate the percentage of mycorrhizal colonization, C. mucugensis subsp. mucugensis roots were blenched in 10% KOH and stained using trypan blue according to the methodology described by Phillips and Hayman (1970). The estimate of colonization of root segments was based on the method of intersection enlarged (McGonigle et al., 1990).
 
Extraction and quantification of spore production
 
Spores of rhizosphere soil samples were extracted following the technique of decanting and wet sieving of Gerdemann and Nicolson (1963) combined with the technique of centrifugation in sucrose solution at 50% of Jenkins (1964). The isolated spores were quantified in a Petri dish and stored in tubes, kept in the refrigerator until analysis of taxonomic characteristics needed for identification.
 
Taxonomic identification of AMFs
 
The spores were previously isolated in separate groups of morphotypes under a stereomicroscope and then mounted on slides with permanent PVLG resin and Melzer reagent (Morton et al., 1996). Spores preserved on slides were observed under an optical microscope (magnification of 1000x) and morphological characters such as size (in µm), shape, color, structure and decoration of wall, type of hyphae and spore germination mode, were recorded for comparison with the related literature. The identification was carried out by using Schenck and Perez (1988) manual and current avaliable literature.
 
Statistical data analysis
 
The data obtained for rosette dry mass, spore number and percentage of mycorrhizal colonization were compared by a one-way ANOVA/Tukey multiple comparison or a t-test when appropriate. The analyzes were performed in the statistical package STATISTICA 8.0 (Statsoft 2002).


 RESULTS

Of the total 12 sample units for each treatment collected after three months (Figure 1A, B and C) of growth in greenhouse, 100% of C. mucugensis subsp. mucugensis plants inoculated with native AMF and inoculated with AMF plus microbiota filtrate survived. Three plants from microbiota filtrate treatment and four from control died. At nine months of growth plants initiate scape (flowering) production (Figure 1D) and some flowers were obtained at  the  end  of  eleven  month  of  growth  at  greenhouse (Figure 1B). At the end of the experiment from the eight remaining sampling units, five plants from mycorrhiza treatment and seven plants from mycorrhiza plus filtrate treatment survived. On the other hand, seven plants from filtrate treatment and seven plants from control died.
 
 
Aboveground biomass
 
Rosette dry mass from three month growth plants of C. mucugensis subsp. mucugensis presented significant differences (p £ 0.05) among mycorrhiza treatments and non mycorrhizal (control and microbiota filtrate) plants evidenced the strong influence of AMF on biomass production (Table 1). The mean values of rosette biomass of eleven months growth plants from AMF inoculated and AMF plus microbiota filtrate did not statistically differ (t test p≤0.05) because only one plant from control and microbiota filtrate treatments survived; statistical analysis was not carried out, but the diference from mycorrhiza treatments was evident (Table 1).
 
 
Mycorrhizal colonization
 
The   mean  values  of  mycorrhizal  colonization  in  AMF inoculated and AMF plus filtrate plants did not differ significantly from each other in both collection times; however, there was an increase in the percentage of colonization of these two treatments when comparing the three and eleven months palnt (Table 1). AMF inoculated plants showed the highest percentages of colonization in root fragments of plants evaluated at three and eleven months of growth. In roots of non-inoculated control and microbiota filtrate inoculated plants, no signal of mycorrhizal structures were observed in both periods (Table 1). During qualitative evaluation with microscope, intraradical hyphae (Figure 2A) and vesicles (Figure 2B) were observed, however arbuscules were the structures more frequently observed (Figure 2C and D).
 
 
Nutritional diagnosis
 
The levels of macro and micronutrients observed in composed samples of rosette dry biomass C. mucugensis subsp. mucugensis at three and eleven months of growth are presented in Table 2. The yield of dry matter of filtrate and control plants in eleven months old plants was insufficient for chemical analysis, therefore are not presented in Table 2. In general, there was no large   variation   on   nutrient  levels  among  plants  from differet treatments.
 
 
Quantification of spores
 
The evaluation of the number of AMF spores of soil rhizosphere demonstrated, as expected,  mycorrhiza  and
mycorrhiza plus microbiota filtrate plants presented significat differences when compared with filtrate and control plants, but not significately different between them (Table 1).
 
Quantification performed for eleven month old plants presented mean values not statiticaly different between filtrate plus mycorrhiza and mycorrhiza plants (Table 1). 
 
 
Statistical analysis was not performed on the control and filtrate plants due to death of plants.
 
Taxonomic identification of AMFs
 
Spores isolated from rhizosphere soil from plants of C. mucugensis subsp. mucugensis inoculated with mycorrhizae and mycorrhiza plus filtrate used to identify seven species of AMFs listed below:
 
1. Claroideoglomus etunicatum (W.N. Becker & Gerd.) C. Walker & A. Schüßlera
2. Glomus macrocarpum Tulasne & Tulasne
3. Glomus microaggregatum Koske, Gemma & Olexia
4. Glomus microcarpum Tulasne & Tulasne
5. Glomus sp.
6. Scutellospora dispurpurascens J.B.Morton & Koske
7. Scutellospora spiniosissima C.Walker & Cuenca
 
The Claroideoglomus etunicatum and Glomus macrocarpon species were the only species found in both treatments. These spores are shown in Figure 3.
 


 DISCUSSION

High rates of root colonization by native AMF was observed in C. mucugensis subsp. mucugensis micropropagated plants. These rates influenced growth responses of plants and showed the mycorrhizal dependence (mycotrophism) of C. mucugensis subsp. mucugensis since non-inoculated AMF plants, even with frequent nutrient solution fertilization on the natural soil, did not grow but died. Our results clearly pointed that C. mucugensis subsp. mucugensis is a mycotrophic plant with rate of mycorrhizal colonization of eleven old months higher than those observed by Pagano and Scotti (2009) on Paepalanthus bromelioides and Aristizabal et al. (2004) in roots of Paepalanthus sp., two Eriocaulaceae species. This rate of colonization by AMFs is also seen in other studies with plants of semi-arid environments (with low  water  availability)  which  showed  a  high  symbiotic effectiveness between AMF and plant species (Yamato et al., 2008; Estrada et al., 2013).
 
The effectiveness of the symbiosis between the micropropagated plants of C. mucugensis subsp. mucugensis and native AMFs was also verified by the production of extensive arbuscules, hypha and spores (completing life cycle of the fungus). Spore density in soil of three-month-old mycorrhized plants of C. mucugensis subsp. mucugensis growth at greenhouse was similar to those observed by Borba and Amorim (2007) in rhizosphere soil (1014 spores 100 g -1 soil) from natural plant population of same plant collected in Mucuge. Number of spores observed in mycorrhizal plants of C. mucugensis subsp. mucugensis can be considered high, demonstrating the dependence of this plant species on AMF for their development. Pagano and Scotti (2009) studying Paepalanthus bromelioides reported 139 spores per 100 g of rhizosphere sandy soil collected from field.
 
It was possible to isolate and identify seven species of native AMF from mycorrhizal plants of C. mucugensis subsp. mucugensis, and with the exception of Scutellospora spiniosissima, all other AMF identified were reported in massive study of Carvalho et al. (2012) that identified and listed 49 species of AMFs collected in rupestrian field of Minas Gerais.
Nutrient analyses of C. mucugenis var. mucugensis rosette demonstrated that mycorrhizal plants presented concentration of macro and micronutrients similar to those non-AMF inoculated plants, despite markedly difference in the plant growth. As known, probably, this is the first report on nutrient staus of a Eriocaulaceae plant, so, it is difficult to compare nutrients concentration on leaves of C. mucugenis var. mucugensis micropropaged plants with other poales plants for example. When we compare leaf nutrients between plants collected on field and from greenhouse experiment, it is observed that concentration of some nutrients such as N and P were higher (three-fold and ten-fold, respectively) in greenhouse plants than field collected plants due to frequent irrigation with nutrient solution.
 
The presence of DSF in the roots of C. mucugensis subsp. mucugensis observed in AMF treatments possibly occurred  during  inoculation,  the same being adhered to AMFs spores were isolated from soil samples. Reports of the coexistence of DSFs and AMFs in the roots of plants stressed environments (arid environments, acidic and nutrient-poor soils) have become increasingly common in studies involving symbiotic associations with fungi (Lingfei et al., 2005; Porras-Alfero et al., 2008; Schmidt et al., 2008).
 
The filtrate of soil microorganisms combined with the native AMFs also had favorable responses on survival and acquisition of dry matter of micropropagated C. mucugensis subsp. mucugensis plants. However, when inoculated alone, microbiota filtrate did not promote plant growth and reduced the plant survival as observed in the control plants. The influence of soil microbiota on plant development as well as possible interactions between the microbial communities present in the rhizosphere and their consequent contribution to plant productivity are widely discussed in the literature (Walker et al., 2003; Artursson et al., 2006; Bonfante and Anca, 2009; Smith and Smith, 2011).
 
Native AMFs inoculated in C. mucugenis var. mucugensis were essential for plants survival and growth, permitting the acclimatization at greenhouse on natural soil. The establishment of in vitro grown seedlings in soil is hampered by weak root system at the beginning of acclimation, however, the symbiotic association between AMF and plant roots increases the survival rate of plant to strengthen the root system (Yadav et al., 2012). This strengthening can reflect the importance of AMF for nutrients and water uptake at low fertilized environments, defense against pathogens, decreased water stress improving some important characteristics for plant acclimation (Joshee et al., 2007; Pindi, 2011; Singh et al., 2012; Yadav et al., 2013).
 
In this study, a relatively high amount of organic matter was observed in the soil (76 g dm−3), one of soil characteristic that may have influenced the number of AMF species found. Borba and Amorim (2007) justified the increased number of species of mycorrhizal fungi in the rhizosphere soil, possibly due to a greater accumulation of soil organic matter. Moreover, the species richness from the rhizosphere soil of potted C. mucugensis subsp. mucugensis may have been influenced by soil type and growing conditions. According to Carvalho (2012), the high diversity of AMF on rupestrian fields can be explained by the heterogeneity of habitats in this environment and the occurrence of AMF species influenced by soil physical properties and also tolerance of these species to low humidity, as shown in some quantitative studies (Conceição and Pirani, 2005).


 CONCLUSION

In this study, the authors reported on native AMF populations inoculated on C. mucugensis subsp. mucugensis plants, but the influence of one determined fungi species was not tested and is a subsequent step  to evaluate the influence of mycorrhiza inoculation. The study shows that AMF inoculation is undoubtedly an important biotechnological tool and encourages the use of these microorganisms in conservation programs of endangered C. mucugensis subsp. mucugensis.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interest.

 



 REFERENCES

Aristizabal C, Rivera EL, Janos DP (2004). Arbuscular mycorrhizal fungi colonize decomposing leaves of Myrica parvifolia, M. pubescens and Paepalanthus sp. Mycorrhiza 14:221-228.
Crossref

 

Artursson V, Finlay RD, Jansson JK (2006) Interactions between arbuscular mycorrhizal fungi and bacteria and their potential for stimulating plant growth. Environ. Microbiol. 8:1-10.
Crossref

 
 

Bonfante P, Anca LA (2009). Plants, mycorrhizal fungi, and bacteria: a network of interactions. Ann. Rev. Microbiol. 63:363-383.
Crossref

 
 

Borba MF, Amorim SMC (2007). Fungos micorrízicos arbusculares em sempre-vivas: subsídio para cultivo e replantio em áreas degradadas. Rev. Biol. Ciênc. Terra 7:2.

 
 

Borkowska B (2002). Growth and photosynthetic activity of micropropagated strawberry plants inoculated with endomycorrhizal fungi (AMF) and growing under drought stress. Acta Physiol. Plant 24:365-370.
Crossref

 
 

Carvalho F, Souza FA, Carrenho R, Moreira FMS, Jesus EC, Fernandes GW (2012). The mosaic of habitats in the high-altitude Brazilian rupestrian fields is a hotspot for arbuscular mycorrhizal fungi. Appl. Soil Ecol. 52:9-19.
Crossref

 
 

Cerqueira CO, Funch LS, Borba EL (2008). Fenologia de Syngonanthus mucugensis Giul. subsp. mucugensis e S. curralensis Moldenke (Eriocaulaceae), nos municípios de Mucugê e Morro do Chapéu, Chapada Diamantina, BA, Brasil. Acta Bot. Bras. 22:962-969.
Crossref

 
 

Conceição AA, Pirani JR (2005). Delimitação de habitats em campos rupestres na Chapada Diamantina: substratos, composição florística e aspectos estruturais. Bol. Bot. Univ. São Paulo 23:85-111.

 
 

Costa FN, Trovó M, Sano PT (2008). Eriocaulaceae na Cadeia do Espinhaço: riqueza, endemismos e ameaças. Megadiversidade 4:117-125.

 
 

Echternacht L, Trovó M, Sano PT (2010) Rediscoveries in Eriocaulaceae: seven narrowly distributed taxa from the Espinhaço Range in Minas Gerais, Brazil. Feddes Repert. pp. 121:3-4, 117-126.
Crossref

 
 

Estrada B, Aroca R, Azcón-Aguila C, Barea JM, Ruiz-Lozano JM (2013). Importance of native arbuscular mycorrhizal inoculation in the halophyte Asteriscus maritimus for successful establishment and growth under saline conditions. Plant Soil 370:175-185.
Crossref

 
 

Gerdemann JW, Nicolson TH (1963). Spores of mycorrhizal endogone species extracted from soil by wet sieving and decanting. Trans. Br. Mycol. Soc. 46:235-244.
Crossref

 
 

Giulietti AM, Hensold N (1990). Padrões de distribuição geográfica dos gêneros de Eriocaulaceae. Acta Bot. Bras. 4:133-151.
Crossref

 
 

Giulietti AM, Harley RM, Queiroz LP, Wanderley MGL, Van Den Berg C (2005). Biodiversidade e conservação das plantas no Brasil. Megadiversidade 1:52-61.

 
 

Giulietti N, Giulietti AM, Pirani JR, Menezes NL (1988). Estudos em sempre-vivas: importância econômica do extrativismo em Minas Gerais, Brasil. Acta Bot. Bras. 1:179-193.
Crossref

 
 

Hazard C, Gosling P, van der Gast JC, Mitchell DT, Doohan FM, Bending GD (2013). The role of local environment and geographical distance in determining community composition of arbuscular mycorrhizal fungi at the landscape scale. ISME J. 7:498-508.
Crossref

 
 

Hoagland DR, Arno DI (1950). The water culture method of growing plants without soil. Berkeley: University of California/College of Agriculture/Agricultural Experiment Station 32:347.

 
 

Jaizme-Vega MC, Azcón R (1995). Effec of vesicular-arbuscular mycorrhizal fungi on pineapple [Ananas comosus (L.) Merr.] in the Canary Islands. Fruits Paris 46:47-50.

 
 

Jenkins WRA (1964). Rapid centrifugal-flotation technique for separating nematodes from soil. Plant Dis. Report 48:692-694.

 
 

Joshee N, Mentreddy SR, Yadav AK (2007). Mycorrhizal fungi and growth and development of micropropagated Scutellaria integrifolia plants. Ind. Crops Prod. 25:169-177.
Crossref

 
 

Kapoor R, Sharma D, Bhatnagar AK (2008). Arbuscular mycorrhizae in micropropagation systems and their potential applications. Sci. Hortic. 116:227-239.
Crossref

 
 

Lima-Brito A, Resende SV, Alvim BM, Carneiro CE, Santana JRF (2011). In vitro Morphogenesis of Syngonanthus mucugensis Giul. Subsp. Mucugensis. Ciênc. Agrotecnol. 3:502-510.
Crossref

 
 

Lima-Brito A, Albuquerque MMS, Sheila VR, Carneiro CE, Santana JRF (2016). Rustificação in vitro em diferentes ambientes e aclimatização de microplantas de Comanthera mucugensis Giul. subsp. mucugensis. Rev. Ciênc. Agron. 47:152-161.

 
 

Lingfei L, Anna Y, Zhiwei Z (2005) Seasonality of arbuscular mycorrhizal symbiosis and dark septate endophytes in a grassland site in southwest China. FEMS Microbiol. Ecol. 54:367-373.
Crossref

 
 

Mcgonigle TP, Miller MH, Evans DG, Fairchild GL, Swana JA (1990). New method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol. 115:495-501.
Crossref

 
 

MMA (Ministério do Meio Ambiente) (2008). Lista Oficial das Espécies da Flora Brasileira Ameaçadas de Extinção. 

View.

 
 

MMA/PNMA (Ministério do Meio Ambiente/ Política Nacional do Meio Ambiente) (1996). Projeto Sempre Viva: Plano de Manejo. Programa Nacional de Infra-Estrutura Turística P 103.

 
 

Moreira BC, Mendes FC, Mendes IR, Paula TA, Prates Jr P, Salomão LCC, Stumer SL, Otoni WC, Guarçoni MA, Kasuya MCM (2015). The interaction between arbuscular mycorrhizal fungi and Piriformospora indica improves the growth and nutrient uptake in micropropagation-derived pineapple plantlets. Sci. Hortic. 14:183-192.
Crossref

 
 

Pagano MC, Scotti MR (2009). A survey of the mycorrhiza occurrence in Paepalanthus bromelioids and Bulbostylis sp. in rupestrin, Brazil. Micol. Appl. Int. 21:1-10.

 
 

Paixão-Santos J, Dornelles ALC, Silva JRS, Rios AP (2003). Germinação in vitro de Syngonanthus mucugensis giulietti. Sitientibus série Ciênc. Biol. 3:120-124.

 
 

Pêgo RG, Paiva PDO, Paiva R (2013). Micropropagation of Syngonanthus elegantulus. Ciênc. Agrotecnol. Lavras 37:32-39.

 
 

Phillips JM, Hayman DS (1970). Improved procedures for clearing roots for rapid assessment of infection. Trans. Brit. Mycol. Soc. 55:158-161.
Crossref

 
 

Porras-Alfaro A, Herrera J, Sinsabaugh RL, Odenbach JR, Lowrey T, Nativie DO (2008). Novel root fungal consortium associated with a dominant desert grass. Appl. Environ. Microbiol. 74:2805-2813.
Crossref

 
 

Raij B, Andrade JC, Cantarella H, Quaggio JÁ (2001). Análise química para avaliação da fertilidade de solos tropicais. Campinas Inst. Agron. 285p.

 
 

Ramos COC, Borba EL, Funch LS (2005). Pollination in Brazilian Syngonanthus (Eriocaulaceae) Species: Evidence for Entomophily Instead of Anemophily. Ann. Bot. 96:387-397.
Crossref

 
 

Rocha FS, Sagin Jr OJ, Silva EMR, Lima WL (2006). Dependência eresposta de mudas de cedro a fungos micorrízicos arbusculares. Pesqui. Agropecu. Trop. 41:77-84.
Crossref

 
 

Sano PT (2004). Actinocephalus (KÖRN.) SANO (Paepalanthus sect. Actinocephalus), a new genus of Eriocaulaceae, and other taxonomic and nomenclatural changes involving Paepalanthus MART. Taxon 53:99-107.
Crossref

 
 

Schenck NC, Pérez Y (1988). Manual for the identification of VA mycorrhizal fungi, 2nd edn. Gainesville, IFAS, University of Florida pp. 18-214.

 
 

Schmidt SK, Sobieniak-Wiseman LC, Kageyama SA, Halloy SRP, Schadt CW (2008). Mycorrhizal and dark-septate fungi in plant roots above 4270 meters elevation in the Andes and Rocky Mountains. Arctic Antarctic Alpine 40:576-583.
Crossref

 
 

Singh NV, Singh SK, Singh AK, Meshram DT, Suroshe SS, Mishra DC (2012). Arbuscular mycorrhiral fungi (AMF) induced hardening of micropropagated pomegranate (Punica granatum L.) plantlets. Sci. Hortic. 136:122-127.
Crossref

 
 

Smith SE, Smith FA (2011). Roles of Arbuscular Mycorrhizas in Plant Nutrition and Growth: New Paradigms from Cellular to Ecosystem Scales. Ann. Rev. Plant Biol. 62:227-250.
Crossref

 
 

Stancato GC, Silveira APD (2010). Micorrização e adubação de mudas micropropagadas de antúrio, cv. Eidibel: crescimento e aclimatização ex vitro. Bragantia 69:957-963.
Crossref

 
 

Sudová R, Vosátka M (2008). Effects of inoculation with native arbuscular mycorrhizal fungi on clonal growth of Potentilla reptans and Fragaria moschata (Rosaceae). Plant Soil 308:55-67.
Crossref

 
 

Villarreal TC, Medina ME, Ulloa SM, Darwin RO, Bangeppagari M, Selvaraj T, Sikandar IM (2016). Effect of Arbuscular mycorrhizal fungi (AMF) and Azospirillum on growth and nutrition of banana plantlets during acclimatization phase. J. Appl. Pharmaceut. Sci. 6:06.

 
 

Walker TS, Bais HP, Grotewold E, Vivanco JM (2003). Root exudation and rhizosphere biology. Plant Physiol. 132:44-51.
Crossref

 
 

Yadav K, Aggarwal A, Singh N (2013). Arbuscular mycorrhizal fungi (AMF) induced acclimatization, growth enhancement and colchicine contento f micropropagated Gloriosa superba L. plantlets. Ind. Crops Prod. 45:88-93.
Crossref

 
 

Yadav K, Singh N, Aggarwal A (2012). Arbuscular Mycorrhizal Technology for the Growth Enhancement of Micropropagated Spilanthes acmella Murr. Plant Protect Sci. 48:31-36.

 
 

Yamato M, Ikeda S, Iwase K (2008). Community of arbuscular mycorrhizal fungi in a coastal vegetation on Okinawa island and effect of the isolated fungi on growth of sorghum under salt-treated conditions. Mycorrhiza 18:241-249.
Crossref

 
 

Zemke JM, Pereira F, Lovato, PE, Silva AL (2003) Avaliação de substratos para inoculação micorrízica e aclimatização de dois porta-enxertos de videira micropropagados. Pesqui. Agropecu. Bras. 38:1309-1315.
Crossref

 

 




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