Vol. 9(16), pp. 1244-1251, 17 April, 2014 DOI: 10.5897/AJAR2013.8025 Article Number: F5B34FF43959 African Journal of Agricultural ISSN 1991-637X Copyright © 2014 Research Author(s) retain the copyright of this article http://www.academicjournals.org/AJAR Full Length Research Paper Spore density and diversity of Arbuscular mycorrhizal fungi in medicinal and seasoning plants Regine Cristina Urcoviche1, Murilo Castelli1, Régio Márcio Toesca Gimenes2 and Odair Alberton1,2* 1Biotechnology Applied to Agriculture, Paranaense University – UNIPAR, Umuarama, Paraná, Brazil. 2Paranaense University – UNIPAR, Umuarama, Paraná, Brazil. Received 3 October, 2013:Accepted 20 February, 2014 Arbuscular mycorrhizal fungi (AMF) set mutualistic symbiosis with most plants. Understanding this association and meet the diversity of AMF in both the medicinal and the seasoning herbs is very important, since these plants have increasingly contributed to improving the quality of human life. The aim of this study was to assess the spore density, taxonomic diversity, and root colonization by AMF in experimental beds of rosemary (Rosmarinus officinalis L.), nasturtiums (Tropaeolum majus), mint (Mentha crispa L.), boldo (Peumus boldus), oregano (Origanum vulgare) and chamomile (Matricaria chamomilla), all planted in the Medicinal Plant Nursery of the Paranaense University - UNIPAR, Umuarama – PR. Soil samples (0 to 10 cm depth) and plant roots were collected in two periods, June and November 2011. Colonization of plant roots by AMF ranged 17 to 48%. The rosemary treatment was highly responsive to the sampling periods, with only 17% of root colonization in June compared with 48% in November. The AMF spore density was higher in June than in November for all species of plants studied. Among the AMF identified within this study, the dominant genus was Glomus sp., followed by Acaulospora sp. in all plants analyzed. Greater knowledge over diversity and density of AMF spores can strongly contribute to the sustainable management of nutrition for medicinal and seasoning plants, particularly on phosphorus supply. Key words: Diversity of mycorrhizal fungi, symbiosis, mycorrhizae, medicinal and seasoning plants. INTRODUCTION Arbuscular mycorrhizal fungi (AMF) compose a key from the excessive use of agrochemicals through functional group of the soil biota that can substantially conventional farming practices (Gianinazzi et al., 2010). contribute to plant yields and ecosystem sustainability in The AMF are commonly found in nature and very crop production strategies. Presently, applications of important as biofertilizers. They belong to Phylum beneficial microbial inoculants (biofertilizers) are Glomeromycota, Class Glomeromycetes and form a increasingly attracting attention toward sustainable monophyletic group of fungi classified into four orders, agriculture and life quality as a consequence of the need thirteen families, and nineteen genera, with somewhat to solve health and environmental problems resulting 215 species already described (Siqueira et al., 2010). Corresponding author. E-mail: [email protected], [email protected]. Tel: +55 (44) 36212828. Fax: +55 (44) 36212830. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Urcoviche et al. 1245 AMF form mutualistic symbiosis with the roots of most points of each bed (n = 9), washed in water, placed in flasks with plants. Through this symbiosis, the fungus gets preserving solution containing ethyl alcohol, acetic acid and carbohydrates and other elements essential to their formaldehyde (1:1:1) and stored in a refrigerator (4°C) (Souza, 2000) until laboratory analyzes for determining the percentage of development from the host plant, forming new spores by AMF root colonization. sporulation processes. In contrast, the host plant obtains from the soil, with the help of the fungus, water and inorganic nutrients such as phosphorus (P), benefits by Spore density of arbuscular mycorrhizal fungal getting long and bulky roots, and acquires resistance to The spores were extracted from 50 g of soil subsamples using the pathogens and abiotic stress, such as the presence of wet sieving method (Gerdemann and Nicolson, 1963). Each sample heavy metals and water shortage (Carrenho et al., 2007; was suspended in 1 L of water and agitated in a beaker, kept at rest Smith and Read, 2008). for 1 min so that the rougher particles of the soil were decanted, Studies on mycorrhizal symbiosis with medicinal and and then the content was poured on two juxtaposed sieves with seasoning plants are scarce. However, some of these 0.710 mm and 0.053 mm opening; the procedure repeated for four few studies have shown that AMF can increase the times. The material remained at the 0.053 mm sieve was transferred to 50 mL Falcon tubes, centrifuged in distilled water production of secondary compounds containing medicinal (3000 rpm, 3 min), and supernatant discarded. Next, saccharose active ingredients in plants under mycorrhizal symbiosis, solution (50%) was added into the tubes and they were agitated in addition to promoting their growth (Faria et al., 2000; and centrifuged (2000 rpm, 2 min). The spores in the supernatant Russomano et al., 2008). were transferred to the 0.053 mm sieve, washed to eliminate The aim of this study was to assess the content of soil saccharose excess, transferred to Petri dishes and then counted organic matter (SOM), spore density and AMF root under stereoscopic lens (40X). colonization in plants of rosemary (Rosmarinus officinalis L.), nasturtiums (Tropaeolum majus), mint (Mentha crispa Characterization and diversity of AMF L.), boldo (Peumus boldus), oregano (Origanum vulgare) and chamomile (Matricaria chamomilla) cultivated in In Glomeromycota, taxonomy can be performed through experimental beds (plots) in the Medicinal Plant Nursery morphological analysis of the formation, structure and germination of AMF spores. Spores were fixed on semi-permanent slides in two of the Paranaense University - UNIPAR, Umuarama – separate groups: one group with PVLG (polyvinyl alcohol and PR, in two periods, June and November, 2011. glycerol) resin and the other with PVLG resin + Melzer, and counted under a microscope (Morton et al., 1993). The sporocarps were carefully broken and the spores were counted. Species taxa of AMFs were identified using Schenck and Pérez MATERIALS AND METHODS (1988) and INVAM - International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi Experimental field: Soil and root sampling (http://invam.caf.wvu.edu) in addition to other species descriptions. From the number of individuals of each genus, the indexes of Root and soil samples were collected at the Medicinal Plant dominance (Simpson) and diversity (Shannon-Wiener) were Nursery of the Paranaense University - UNIPAR - Campus II, in the estimated according to Souza et al. (2010). They were calculated Umuarama city, northwestern Paraná State at coordinates S 23° according to the equations: 46’ 11.34’’ and WO 53° 16’ 41.78’’. For each plant of rosemary, nasturtiums, mint, boldo, oregano C = -Σ (XI/X0) x log (XI/X0) Simpson, and chamomile were assigned three experimental beds. Roots and 2 rhizosphere soil were sampled in three points of each bed, giving a H’ = -Σ (XI/X0) Shannon-Wiener, total of 9 replications per plant species in a completely randomized design. The beds received organic compost (coffee leaf straw where XI is the spore density of each genus in 100 g of soil, X0 is transformed by fermentation process of composting) before being the total spore density of all AMF genera. planted. Then, as with plants, the beds were irrigated daily by spraying when needed. Plants with the exception of boldo and rosemary, were at the phenological stage of pre-flowering. AMF root colonization The roots and soil sampling was performed at 0 to 10 cm, about 10 cm away from the stem of each plant, into two periods: June and To determine AMF root colonization, six plants with roots were November 2011. Sampling was done at the same point in each bed collected from each subplot (beds) and washed in running water. for the two periods. In each plot, three samples were collected for Plant roots were freshly cut at the length of ±1.5 cm so that they approximately 0.5 kg of soil, placed in plastic bags and stored in a can represented as the whole radicular system. The lab procedure refrigerator (4°C) until laboratory analyzes. was done according to Phillips and Hayman (1970), where sample A soil sample was collected for chemical and granulometric roots are placed in 10% KOH and closed in plastic Falcon Tubes. analyses. One portion of that sample was utilized to determine the After heating the tubes with roots in water bath at 90°C for 1 h, the soil chemical characteristics at Solo Fértil Laboratory in the city of KOH solution was removed and the roots were washed in running Umuarama, Paraná, Brazil. The characteristics determined were: water. A solution of 1% HCL was added in the tubes with roots and 2+ 2+ 3+ –1 pH in CaCl2, Ca , Mg and Al extracted in KCl (1 Mol L ), and P agitated for acidification for 5 min; next, the solution was removed. and K+ extracted in Mehlich-1. All the analyses followed the Then, roots were stained by adding 0.05% trypan blue to the tubes, CELA/PR standards to obtain a greater reliability of the results which were heated in water bath at 90°C for 30 min. At the end of (Table 1). The other portion of the soil sample was intended