بررسی فراوانی قارچ‌های آربوسکولار میکوریزا در خاک و ریشه گیاهان دارویی باغ بوعلی سینا در همدان، ایران

Introduction

Most medicinal plant taxa in the world have been studied for their medicinal values and botanical properties. However, the occurrence of symbiotic fungi in these group of plants was not well studied (1, 2). The cultivation of medicinal herbs will need to learn about soil and plant biology, the environment and agricultural technology. In order to develop an effective method to obtain high quality medicinal materials, the study of symbiotic relationship between arbuscular mycorrhizal fungi (AMF) and medicinal plants is very important. The production and application of AMF biological fertilizers may be effective in the improvement of an actual method in plant production. It may be the most effective method to improve the quality and quantity of the medicinal materials obtained in the non-fertile soils. Arbuscular mycorrhizal fungi can increase plant growth, photosynthesis, nutrients storage, metabolites and beneficial chemical compounds and decrease soil borne plant diseases by inhibition of fungal pathogen (3, 4).

The study of the status of mycorrhizal symbiosis and soil monitoring has a specific value in the medicinal plant cultivation and production in dryland area. Symbiosis of medicinal herbs with AMF can be an effective alternative for improvement of soil-water efficiency by increasing plant tolerance and growth in dry land farming (5-8). Dry and osmotic stresses on plants can be alleviated by various morphological, anatomical, physiological and specially osmoregulation occurring better in plant with mycorrhizal symbiosis (5).

Due to these facts, a preliminary field survey was conducted to examine the mycorrhizal status of medicinal plants collected from the Garden of Medicinal Plants of Bu-Ali Sina in Hamadan in Iran with semiarid climate. The garden has an area of 3.7 h, located in southern part of the city of Hamadan. Where a collection of 213 plant species belong to herbaceous plants (25%), shrubs (41%), trees (27%) and onion plants (7%) from 64 families. In this category, two botanical families, Lamiaceae (39%), and Asteraceae (23%) have more species. Information about the symbiosis of medicinal plant species with AMF in the semi-arid region, allows increasing knowledge of the biology and ecology of these plant species. Our findings on the AMF symbiosis with medicinal plant species would be useful for preparation of AMF bio-fertilizer and application for different plant species in semi-arid regions.

Materials and methods

Study area, soil and plant sampling: This study was down in Bu-Ali Sina Garden of Medicinal Plants in Hamadan city, in northwest of Iran, with semi-arid climate. The minimum and the maximum annual mean temperatures are −8.33 and 40 ^◦C, respectively. The average of annual rainfall and the average of annual temperature are 300 mm and 8.10 ^oC, respectively. The area of this garden is 2.3 hectares. It lies 1870 m higher than see level (48˚32΄30˝ E and 34˚47΄12˝N).

The garden has a rich collection of approximately 250 plant taxa from 48 native and foreign cultured plant families. Selection of annual and perennial herbaceous plant species was mainly on the basis of their growth stage. The sampled and studied plants were 48 species which belong to 9 families (i.e. Lamiaceae with 17 species, Asteraceae 12, Brassicaceae 5, Chenopodiaceae 2, Boraginaceae 2, Malvaceae 2, Zygophyllaceae 1, Plantaginaceae 1, Polygonaceae 1, Euphorbiace 1, Solanaceae 1, Apiaceae 1, Portulacaceae 1 and Fabaceae with 1 species). They were listed in Table 2. Plant roots and root surrounding soils were collected mainly at the flowering stage and early seed formation period in June, 2012. Root systems with soil were excavated intact and transported to the laboratory for analysis. In the case of each taxa, three repetition samples were collected.

Study of root colonization: The roots were prepared and stained according to the modified method described by Giovannetti and Mosse (1980) and were assessed for colonization according to the grid-line intersect method (McGonigle et al., 1990). Briefly, after soil separation and washing in tap water, the roots were putted in 7% KOH for 24 h. Then they were rinsed in water for clearing. The material was acidified in 3.5% HCl (2 h), then stained with 0.05% trypan blue, and finally stored in 50% glycerol. Root fragments (1 cm) were mounted on slides in glycerol. Under light microscope, each intersection for AM fungal structures was evaluated. All of the figures in this paper were presented under light microscope with 400× magnification. There are seven possible, and mutually exclusive categories of intersections: n- no fungal structures, a-arbuscules, v- vesicles, av- arbuscules and vesicles, c- coils, hm- mycorrhizal hyphae (near but not at arbuscules or vesicles), h- hyphae not seen to be connected to arbuscules or vesicles (they may (h1) or may not (h2) belong to AM fungi). Mycorrhizal hyphae are always intersected in a, v, av, c, and hm are known to be mycorrhizal because they are seen to be attached to arbuscules, vesicles, or both. Here 150 intersections were examined for each root sample, where a total of T (n+a+v+av+c+hm+h) intersections were inspected, the percentage of root length colonized by fungi was calculated as:

The percentage of root length colonized by mycorrhizal hyphae = 100 [(a+v+av+c+hm+h1)/T]

Fungal endophytes that accompanied AM fungi in roots, namely dark septate endophytes (DSE) were studied through the assessment of AMF colonization. DSE colonization was identified on the basis of regularly dark pigmented septate hyphae, with occasionally occurring sclerotia (9).

Additionally, the frequency of the occurrence of resting spores of the fungi was assessed in soil around the plant roots.

AMF spores enumeration: Spores were extracted from 50 g soil of each sample by wet sieving followed by floatation-centrifugation in 453.5 g l^-1 sucrose solution (10, 11). The spores were collected from sucrose suspension in beaker on a 25 µ sieve and washed with distilled water to spread spores evenly over the entire of grid pattern filter paper. They were numbered under a stereoscopic microscope (40×). The number of spores was expressed as the mean of three replicates in 10 g soil.

Chemical and physical analyses of soil sampled from around the roots: The chemical and physical analyses of soil were done on air dried and ground (2 mm) samples. Selected soil properties were determined according to standard methods (12, 13). The biological analyses of soil were done on fresh and ground (2 mm) samples stored at 4 ^oC in lab refrigerator. Selected soil biological properties were determined according to standard methods (14). Table 1 shows the studied soil properties and the method applied for each one.

Table 1- The method applied for analysis of soil properties

Statistical analyses: Correlation analyses were performed to evaluate the relationships between different properties of the soil sampled from around the roots, spore number and plant root colonization. All data analyses were performed by excel and correlation analyses were done by SAS, 9.2.

Results

Arbuscules and vesicles in Foeniculum vulgare from Apiaceae family were observed and the percentage of root colonization was calculated 70.51. Arbuscular mycorrhizae with paris type arbuscules were found in this plant species (Table 2).

Arbuscules are the structural and functional criterion of the symbiosis. All of the investigated plant species from Asteraceae had arbuscules and vesicles. They had paris type arbuscular mycorrhizae symbiosis, except Achillea millefolium, Tanacetum parthenium and Arctium lappa which had arum type mycorrhizae symbiosis. Achillea millefolium like other plant species in Asteraceae family had mycorrhiza symbiosis (61.02%) but without vesicle. Although Tanacetum parthenium had no arbuscule and vesicle, the root colonization of this species was also relatively high by fungi (62.96%). But Arctium lappa in this family did not have any arbuscule, vesicle and maycorrhiza symbiosis (fig. 1). The highest root colonization was observed in Chrysanthemum salicornia equal to 77.26%.

Table 2- The sampled and studied plant species from Bu-Ali Sina Garden of Medicinal Plants

# RCP) root colonization percentage, Ves.) vesicle, Arb.) arbuscule, ND) Not detectable, +) visible –) invisible organs

Both sampled plant species from Boraginaceae family had mycorhiza symbiosis (Table 2). The root colonization percentage in Echium amoenum and in Borago officinalis were 38.87 and 43.65 respectively. The type of mycorrhiza symbiosis was paris in these plants. Arbuscules was found in both but vesicles were found only in Borago officinalis root tissues (fig. 2).

Harley and Harley previously reported that from these family roots of Echium vulgare L. can be colonized by VAM. However, Borago officinalis being reported earlier as non-mycorrhizal plant (25, 26). In our study same as Zubec and Blaszkowski (2009) we recognized Borago officinalis as mycorrhizal plant.

Brassica oleracea, Lepidium sativum, Hyoscyamus nicotiana, and Cheiranthus cheiri root tissues did not have any arbuscule, vesicle and mycorrhiza symbiosis. But in root tissues of Eruca sativa from Brassicaceae family we found arbuscule, vesicle and mycorrhiza symbiosis (figs. 3-4). Root colonization percentage in this plant species reached 62.13. Both types of mycorrhiza symbiosis, paris and arum were observed in this plant species.

Beta vulgaris and Spinacia oleracea were sampled and studied from Chenopodiacea family. Harley and Harley reported that Chenopodium album L. can be colonized by AM Fungi. But in many studies this family was named non-mycorrhizal plants. Although they did not have any arbuscule, vesicle and mycorrhiza symbiosis, but dark septate fungi were observed in root tissues of Beta vulgaris frequently (figs. 5-6).

Euphorbia helioscopis from Euphorbiaceae and Securigera securidaca from Fabaceae families both had arbuscule, vesicle and paris type mycorrhiza symbiosis. Root colonization percentage in these plant species were 58.64 and 63.40 respectively.

All of the 17 investigated plant species from Lamiaceae had arbuscular mycorrhizae symbiosis and all of them had vesicles except Stachys lavandulifolia (Table 2). Although arbuscule was not observed clearly in some species, the type of observed fungal hypha was often paris. However, the arum type was observed in root tissues of Thymus kotschyanu. In root tissues of Hyssopus officinalis, Zataria multiflora, Salvia hyderangea and Salvia aethiopis both types of paris and arum mycorrhiza were observed (Fig. 7). The highest root colonization in this family belonged to Teucrium polium equal to 81.99% and the lowest root colonization in this family belonged to Satureja hortensis equal to 32.47%.

In root tissue of Malva sylverstris from Malvaceae family there was not any evidence of mycorrhiza symbiosis (fig. 8), but Althaea officinalis had arbuscule, vesicle and mycorrhiza symbiosis. Root colonization percentage in this plant species reached 63.05.

Plantago major from Plantaginaceae had both arbuscule and vesicle with 78.13% root colonization and paris type symbiosis. Although in root of Rumex asetosella from Poligonaceae was not observed any arbuscule, it had vesicles with 58.64% fungal colonization. Mycorrhiza type in Portulaca oleracea from Portulacaceae was paris. It had 32.33% root colonization without any observable vesicle. Physalis alkekengi from Solanaceae family had both paris and arum type mycorrhiza with 67.02 root colonization without any observable vesicle. Zygophyllum fabago from Zygophyllaceae had no vesicule, arbuscule and mycorrhiza symbiosis (figs. 9-10).

Table 3 shows some important soil properties in mycorrhiza symbiosis. Generally, soil organic carbon was relatively high around the roots of plants with lower mycorrhiza symbiosis. It was higher than 1 g 100 g^-1 soil around the roots of Cnicus benedictus, Lepidium sativum, Beta vulgaris, Spinacia oleracea, and Mentha longifolia. Electrical conductivity was high in soil (>1 dSm^-1) around the roots of Chrysanthemum salicornia, Cnicus benedictus and Securigera securidaca.

The pH of soil sampled from around the roots of plants was not so different. It was between 7.12 and 7.81. Available P in the studied soils was sufficient for plant growth and it was higher than 28.8 mg kg^-1. Available P was higher than 80 mg kg^-1 in soil around the roots of Hyoscyamus nicotiana, Lepidium sativum, Spinacia oleracea, and Zygophyllum fabago. Most of these plants did not have high mycorrhiza symbiosis. This may be related to lower depletion zone around the roots of these plants.

Table 3- The studied Chemical soil properties sampled from around roots of the plant species

The important soil properties in mycorrhiza symbiosis were measured. Table 4 shows the studied biological properties of soil sampled from around the roots of the plant species. Basal respiration in soil sampled from around the roots of Arctium lappa and Borago officinalis was lower than 0.10 mg CO₂ g^‑1 soil day^-1. In contrast, it was higher than 0.20 mg CO₂ g^‑1 soil day^-1 in soil sampled from around the roots of Cnicus benedictus, Brassica oleracea, Lepidium sativum, Beta vulgaris, Spinacia oleracea, Mentha longifolia, Malva sylverstris, Portulaca oleracea, Physalis alkekengi and Zygophyllum fabago. Basal respiration (BR) basically shows simple and active organic carbon but substrate induced respiration (SIR) shows active biomass carbon in soil around the roots. Substrate induced respiration was lower than 1.5 mg CO₂ g^‑1 soil day^-1 in soil around the roots of Arctium lappa, Tanacetum parthenium, Borago officinalis, Echium amoenum, Cheiranthus cheiri, Salvia aethiopis and Physalis alkekengi but it was higher than 1.7 mg CO₂ g^‑1 soil day^-1 in soil around the roots of Brassica oleracea, Lepidium sativum, Beta vulgaris, Spinacia oleracea, Securigera securidaca and Mentha longifolia. Where BR would be high and SIR would be low, it is a hard condition for soil microbiota. So the BR/SIR ratio was calculated for this respect. The highest BR/SIR ratio (>0.15) was obtained in soils around the roots of Cnicus benedictus, Beta vulgaris, Spinacia oleracea and Mentha longifolia. This ratio shows hard physiological condition for soil microorganisms around the root of these plants. However, most of Beta vulgaris, Spinacia oleracea are not good host for AM fungi.

The glumerale spore numbers in soil around the roots of the unpleasant plants for AM fungi were considerably low compared to those numbered in soil around the roots of other plant species. It was lower than 15 spores in 10 g of soil around the roots of Chrysanthemum salicornia, Cnicus benedictus, Brassica oleracea, Lepidium sativum, Beta vulgaris, Salvia hyderangea and Zygophyllum fabago. Most of these plants did not have a high root colonization percentage. Some of them like Brassica oleracea, Lepidium sativum, Beta vulgaris, and Spinacia oleracea were not good photosymbiont for mycorrhiza symbiosis. A large part of the differences between AM fungi spore numbers in soils might be related to their different plant species. Land use and plant diversity can change soil properties, controlling soil microbial population and activities (27). It was showed that abundance and composition of AM fungi community are strongly influenced by the host species and land use through differential effects on hyphal growth and sporulation (28-30). In our pervious study, the highest AMF spore numbers counted in soils sampled from dry farmlands mainly covered with Poaceae. These lands had significantly low fertility and moisture contents.

Table 4- The studied biological soil properties sampled from around roots of the plant species

In contrast, the lowest AMF spore numbered in soils sampled from coniferous woodlands. These differences strongly related to specific plant effects on AM fungi in soil. Root exudates of different species are different, influencing the germination and growth of specific AM fungi species (31, 32). Although AM fungi are non-host-specific and they have ability to infect a wide range of hosts, degree of benefit to each associate in any fungous and plant association can depend on the specific species involved (28, 33).

Totally in the studied soils the activity of alkaline phosphatase was higher than the activity of acid phosphatase. The activity of alkaline phosphatase was considerably high (>20 umol PNP g^-1 soil h^-1) in soil around the roots of Achillea millefolium, Calandula officinalis, Eruca sativa Lam, Lepidium sativum, Rosmarinus officinalis, Satureja hortensis and Zataria multiflora boiss. Except Lepidium sativum, these plant species had relatively high root colonization percentage. Acid phosphatase activity was relatively high in soil around the roots of Achillea millefolium, Aster trapolium, Calandula officinalis, Securigera securidaca, Thymus kotschyanu and Plantago major. It was higher than 9.0 umol PNP g^-1 soil h^-1. These plants were also good photosymbiont for AMF (Table. 1)

Correlation analysis showed that there were positive and significant relationship between basal respiration, substrate induced respiration, organic carbon (OC) and clay contents in the sampled soils. In contrast to those properties, there were positive and significant relationships between soil alkaline and acid phosphatase activities. Acid phosphatase and alkaline phosphatase activities had negative and significant correlation with soil available phosphorus and clay contents respectively. Root colonization and spore numbers were correlated to each other and both had negative and significant correlation with soil available phosphorus (Table. 5). The correlation coefficients between root colonization and soil pH, EC, BR, SIR, BR/SIR ratio, OC, available P, silt, and clay contents were negative. In contrast to those soil properties, acid and alkaline phosphatase activities and also sand content of soil had positive correlation with root colonization and spore numbers in soil. Anyway the correlations of root colonization with soil clay content, available P and acid phosphatase activity was statistically significant. In contrast, the significant of the correlations between spore numbers in soil around the roots of plant and the studied soil properties were more obvious. Same as root colonization, spore number was positively related to soil acid and alkaline phosphatase activities and sand content and negatively related to the soil pH, EC, BR, SIR, BR/SIR ratio, OC, available P, silt, and clay contents. All of the soil properties except acid and alkaline phosphatase activities had significant correlation with soil spore numbers (Table 5). Thus, spore numbers were more related to soil properties and root colonization was more related to plant species.

Here in this garden ecosystem, the studied plant species would be highly dependent on AM associations for survival in the infertile and sandy soils compared to more fertile and clay soil. Several reports have shown that increasing soil fertility especially concentrations of soluble phosphate in soils can decrease fungal colonization (Graham et al., 1981; Asimi et al., 1980; Plenchette et al., 1983; Schwab et al1983; Guillemin et al., 1995). In addition, it was reported that abundance of AMF and their spores depend on physica1 characteristics of soil (Ortega-Larrocca et al., 2001). In this study, the correlations between spore numbers and soil sand, silt and clay contents were significant and the root colonization was related positively to soil sand content and negatively to soil silt, clay, OC and available P. Higher root colonization may be a cause of higher acid and alkaline phosphatases in soil around the roots of these medicine plant (Table 5).

Table 5- Liner relation between root colonization percentage and spore numbers with the studied soil properties