Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of drought stress

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Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of drought stress Sedigheh Gheisari Zardak a , Mohsen Movahhedi Dehnavi a , Amin Salehi a,∗ , Majid Gholamhoseini b a b

Agronomy and Plant Breeding Department, Yasouj University, Yasouj, Iran Oil Seed Crops Department, Seed and Plant Improvement Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

a r t i c l e

i n f o

Article history: Received 22 December 2015 Received in revised form 21 September 2016 Accepted 22 September 2016 Available online xxx Keywords: Arbuscular mycorrhizal fungi Essential oil Fennel Medicinal plants Osmotic adjustment Water deficit stress

a b s t r a c t The influence of colonization of fennel (Foeniculum vulgare Mill.) roots by two different species of arbuscular mycorrhizal (AM) fungi (Glomus mosseae and Glomus intraradices) and different irrigation treatments (well-watered, moderate water deficit stress, severe water deficit stress and very severe water deficit stress) were examined on growth, osmotic adjustment and qualitative and quantitative yield during two consecutive growing seasons. The experiments were conducted at Yasouj University, Yasouj, Iran located in semi-arid region of Iran. The results indicated that irrespective of mycorrhizal species and water deficit stress intensity, inoculated seeds produced taller plants, more dry matter and seed yield as well as seed essential oil content compared with non-inoculated plants. In both years of the experiment and in AM free plots, irrigation treatments showed a significant effect on leaf P concentration so that P concentration decreased with increasing water deficit stress intensity. Interestingly, mycorrhizal inoculation increased P accumulation and soluble sugars in fennel leaves compared with control plants. The data presented in this study suggest that different AM fungi species even within the same genus have distinct effects on fennel growth and yield. Overall, the results showed that G. mosseae was more efficient under water deficit stress. The application of AM fungi could be critical in cultivation of fennel under arid and semi-arid conditions, where water is the most important factor in determining plant growth and yield. © 2016 Published by Elsevier GmbH.

1. Introduction Conventional farming practices negatively affect soil and water quality worldwide. In recent decades, agricultural goods production caused changes in environment functions resulting in unsuitable resource management. It has been reported that irrigated arid and semi-arid areas, where intensive agricultural is practiced, tend to experience problems of water resource deficiency and environmental pollution by excessive irrigation and chemical fertilizer applications (Iqbal et al., 2012; Magesan et al., 2002). Therefore, recent research has focused on new and practical management options for reducing water and nutrient loss, improving soil quality and increasing crop yield (Edmeades, 2003). Organic and low input cropping systems are the most important objectives of sustainable agriculture, so application of biofertilizers to reduce chemical fertilizers use is a big step towards sustainability. Biofertilizer application improves physical, chem-

∗ Corresponding author. E-mail address: [email protected] (A. Salehi).

ical and biological properties of soil and increases soil fertility without destructive effects on the environment. A promising strategy to achieve sustainability in agriculture is application of useful microorganisms, such as arbuscular mycorrhizal (AM) fungi, which have an important role in water and nutrient supply for plants in organic and sustainable agriculture systems (Ishizuka, 1992). AM fungi belong to a group of microorganisms that live in a symbiotic relationship with most of the crops and medicinal plants (Wu and Xia, 2006). These fungi form essential components of sustainable soil-plant systems and improve crop growth and productivity (Goussous and Mohammad, 2009). Water shortage is being intensified by population growth, climate change and bio-fuel crop expansion, leading to concerns about global food security and environmental sustainability (Qiu et al., 2012). Since agriculture is the largest water consuming sector of the economy, it should be considered as an important part of solution (Jing et al., 2012). On the other hand, water is one of the major limiting factors affecting plant growth and development, especially in arid and semi-arid regions, where plants are often exposed to periods of water shortage. Drought stress is an important cause of crop losses worldwide, reducing average yield by more than 50%

http://dx.doi.org/10.1016/j.jarmap.2016.09.004 2214-7861/© 2016 Published by Elsevier GmbH.

Please cite this article in press as: Gheisari Zardak, S., et al., Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of drought stress. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.09.004

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Precipitation 2012 Temperature 2011

350

30

300

25

250

20

200

15

150

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100

Temperature ( o C)

(Wang et al., 2003). Fortunately, AM fungi can mitigate adverse effects of drought stress in plants. AM fungi are able to produce a very extensive network of hyphae in the soil when in symbiosis with host plants. During the formation of arbuscular mycorrhizae, fungal hyphae enter the epidermal, exodermal and cortical cell layers of the roots, reaching the inner cortex, where the functional units, the arbuscules, develop (Gholamhoseini et al., 2013). The fungi also form hyphae outside of the plants, extending the root–soil interface to facilitate nutrients and water uptake (Kistner and Parniske, 2002). AM fungi enable host plants to establish itself and grow more efficiently, even under biotic and abiotic stress conditions (Porcel and Ruiz-Lozano, 2004), through a series of complex communications between the host and the fungus (Harrier, 2001). There are numerous reports of fungal symbionts conferring host plant tolerance to various stresses, including drought, heat, salt, metals, and even diseases (Waller et al., 2005; Márquez et al., 2007; Rodriguez et al., 2008). Therefore, the symbiotic interactions of plants with mycorrhizal fungi are agriculturally and ecologically important (Indrasumunar, 2007). In addition, species-specific interactions between host plant and fungal pathogens highlight the importance of screening of different associations to maximize the benefits of the symbiosis (Miller et al., 1987). Considering the physiological differences within species and even within geographic isolates (Bethlenfalvay et al., 1989), the biodiversity of AM is great. There is little information about the physiological specialization and functioning of AM. In fact, AM fungi species differ in their tolerance and in their ability to adapt to environmental conditions (Sylvia and Williams, 1992). The variation within AM species and also different symbiotic strategies occurring in response to water deficit stress, as well as the compatibility with different environmental conditions, suggest that it may be likely to choose the most effective species. Moreover, mycorrhizal colonization has been shown to increase water deficit tolerance in many crops such as corn (Subramanian et al., 2006), wheat (Bryla and Duniway, 1997), soybean (Bethlenfalvay et al., 1988), onion (Azcón et al., 1996) and lettuce (Tobar et al., 1994; Azcón et al., 1996). However, the majority of these experiments have been conducted under controlled conditions like growth chambers or greenhouses. In addition, there is little information on the use of different mycorrhizal fungi species under field conditions to improve medicinal plant yield and quality, especially in semi-arid regions. Although medicinal plants have been used since Biblical times, the interest in essential oils increased during the past decades. Today, the traditional crops are not the only plants used in agricultural systems, medicinal plants whose essential oils are valued for their characteristic aromatic or therapeutic attributes. Increase in demand of pharmaceutical factories for primary materials and conservation of natural genetic resources, enhances the importance of the research and production of medicinal plants. Fennel (Foeniculum vulgare Mill.), as an important medicinal plant, is a member of the Apiaceae family. It is an herbaceous perennial plant originated from Mediterranean regions (Nourimand et al., 2012). Fennel is an aromatic and flavorful plant with culinary and medicinal use. Fennel seeds are anise like in aroma and are used in baking, meat and fish dishes, ice cream, alcoholic beverages and herb mixtures (Diaaz-Maroto et al., 2005). The fennel bulb, leaves and seeds are extensively used in many of the culinary traditions across the world. The major components of fennel seed essential oil are trans-anethole, fenchone, estragol (methyl chavicol), and ␣-phellandrene. The concentration of the compounds depends on phenological stage and origin of the plant (Diaaz-Maroto et al., 2006; Omidbeigy, 2000). Fennel is broadly used around the world as mouth fresheners, toothpastes, desserts, antacids and in various culinary applications. Although, more attention has been paid to this plant in the world

Precipitation (mm)

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50 0

0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Month

Fig. 1. Monthly temperature and precipitation during the growing seasons in 2011 and 2012.

and its cultivation area has been increased four times during past two decades, there is still little information on the use of different species of mycorrhizal fungi under field conditions to improve fennel yield and quality in semi-arid regions. This study investigates two different species of mycorrhizal fungi, in terms of their efficiency under four different irrigation regimes, by quantifying fennel growth, yield, and essential oil percentages and yield. 2. Material and methods 2.1. Experiment location and climatic characteristics Field experiments were conducted at the Faculty of Agriculture, Yasouj university, Iran (30◦ 38 N and, 51◦ 32 E, altitude 1832 m), during the 2011 and 2012 growing seasons. The region is characterized as semi-arid, with mean annual precipitation of 86 mm, which mostly falls during the autumn and winter months. The annual mean temperature was recorded 15 ◦ C. The average precipitation and temperature in 2011 and 2012 were similar to the long-term meteorological data trend (Fig. 1). The field was kept fallow during the previous year to reduce the endogenous mycorrhizal fungi population and eliminate their propagules, and decomposition crop residue remained from the previous year. 2.2. Soil sampling and land preparation Prior to the beginning of the experiment, a composite soil sample was collected at the depths of 0–30 cm, and the air-dried, crushed and tested for various physical and chemical properties. The soil type was found to be a clay loam with 0.19% total N, 348 ppm available K, 15 ppm available P, EC = 0.8 ds m−1 , pH = 7.5, water content at field capacity (FC) = 35% volumetric moisture and water content in permanent wilting point (PWP) = 15% volumetric moisture. In addition, the soil was evaluated in terms of biological factors for this purpose a wet-sieving technique was used to extract spores, and the most probable number (MPN) test was used to determine the number of propagules (kg−1 ) in the soil. Since the number of extracted propagules from the soil was extremely low (2–3 kg−1 ), no attempt was made to fumigate the soil before applying the treatments. Experimental plots were prepared after plowing and diskharrowing. The plots were 5 m long and consisted of six rows, 50 cm apart. There were 2.5 m gaps between the blocks, and 1.5 m alley was established between the plots to prevent lateral water movement and other interferences. The mycorrhizal fungal inoculants consisted of spores and hyphal root fragments from stock cultures of Glomus mosseae and Glomus intraradices. The dose of inocula was 80 kg ha−1 . The G. mosseae and G. intraradices inocula were selected because of their commercial availability in Iran and also in

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Table 1 Analysis of variance for the effects of different treatments on the measured traits. Source of Variation

df

a

PH

UNPP

GNPN

LPC

LSSC

DMY

SY

EOP

EOY

Replication Irrigation Error a Mycorrhiza Irrigation × Mycorrhiza Error b Year Year × Irrigation Year × Mycorrhiza Year × Irrigation × Mycorrhiza C.V (%)

3 3 9 2 6 24 1 3 2 6

* ** 50.3 ** * 29.73 ** * ** NS 4.75

NS ** 56.13 ** ** 40.9 ** NS NS ** 11.91

* * 223.6 * * 161.4 ** * * ** 17.96

** ** 11.41 ** ** 8.17 ** ** ** ** 13.44

* ** 77.19 ** ** 32.8 ** ** ** ** 2.59

* ** 1218418 * NS 903560 * * * * 9.23

** ** 39570 ** ** 30973 ** ** ** ** 8.45

NS ** 0.193 * NS 0.168 ** NS NS NS 13.07

* ** 90.46 ** ** 39.71 ** ** ** ** 8.12

NS: not significant. * and **: significant at the 0.05 and 0.01 probability level, respectively. a PH: plant height; UNPP: umbel number per plant; GNPN: grain number per umbel; LPC: leaf P concentration; LSSC: leaf soluble sugar content; DMY: dry matter yield; SY: seed yield; EOP: essential oil percentage; EOY: essential oil yield.

the world. The fennel seeds (Foeniculum vulgare Mill. ssp. vulgare, var. vulgare, Hamedan accession) were inoculated with AM fungal inoculum at 200 propagules (Most Probable number, MPN method described by Feldmann and Idczak, 1992) per seed (Miransari et al., 2006; Samarbakhsh et al., 2009) and soaked in water for 24–30 h before sowing to increase germination rate. The seeds were sown during the last week of March in both years. The distance between the plants in the rows was 20 cm; thus, the plant density was approximately 10 plants per m2 . Immediately after sowing, the soil was irrigated. The irrigation cycle of each plot was closed to avoid run off. Irrigation was performed using furrow method and irrigation scheduling was determined according to daily changes of soil water content (SW) at the depth of root development (40 cm depth). A deficit approach was used to estimate irrigation requirements: soil water content at FC was defined as no water deficit. Soil available water was determined by calculating the difference between the water content at FC and PWP (the soil moisture at FC and PWP were determined at 0.1 bar and 1.0 bar, respectively using pressure plate (extractor) apparatus (Obi, 1974)). Until the beginning of umbel formation, irrigation was applied similarly in all the plots when 20% of available water had been consumed at the depth of root development. Upon 10% umbel formation stage, the plots received different irrigation treatments. Time-domain reflectometry (TDR) using probes and access tube (TRIME-FM, England) were used to measure soil water content ( v ) in experimental plots at the depths of 0–40 cm (at 0.1 m intervals). Data on soil volumetric water content were collected daily during the growing seasons. In the first year and before seed sowing, phosphorous fertilizer (from ammonium phosphate source) was applied at the total of 250 kg ha−1 then incorporated into the soil. In both years, nitrogen fertilizer (150 kg ha−1 from urea source) was applied as top dressing at sowing time and stem elongation stages.

2.3. Treatments and data collecting The experiment was conducted using a randomized complete block design with a split plot arrangement of treatments with four replicates. The first factor included four irrigation regimes (I1 : irrigation was initiated after using 20% of the available water (wellwatered), I2 : irrigation was initiated after using 40% of the available water (moderate drought stress), I3 : irrigation was initiated after using 60% of the available water (severe drought stress) and I4 : irrigation was initiated after using 80% of the available water (very severe drought stress)) as main plot, and the second factor included three mycorrhizal fungus treatments (non-inoculation with AM fungi, inoculation with Glomus mosseae species and inoculation with Glomus intraradices species) as sub plot. Weed control was carried out by hand weeding during growing seasons. To deter-

mine dry matter weight (the yield of the whole plants without roots), morphological traits (including plant height measured by ruler), yield components (including umbel number per plant and grain number per umbel measured by counting) and seed yield (measured by weighing), 30 plants from each plot were handharvested and air-dried under shading conditions at physiological maturity stage (when 65–75% of fruits turned brown). At the end of seed filling stage, leaf samples (10 fully expanded leaves from each plot) were taken to determine the P content. Phosphorus concentration in the leaves was measured based on calorimetrically method (using a 6505 JenWay spectrophotometer). In addition, leaf soluble sugar was determined by the anthrone method (Li, 2000) using sucrose as the standard. Half a gram of fresh samples was placed in a 25 mL cuvette added with 10 mL distilled water, allowed to stand at 100 ◦ C for 1 h, and filtered into 25 mL volumetric flasks. Reaction mixture of 7.5 mL contained 0.5 mL extracts, 0.5 mL mixed reagent (1 g anthrone + 50 mL ethyl acetate), 5 mL H2 SO4 (98%), 1.5 mL distilled water. The mixture was heated at 100 ◦ C for 1 min and absorbance read at 630 nm (Wu and Xia, 2006). In order to determine essential oil percentage, 30 g samples were extracted with 300 mL water. To extract essential oils, distillation method with water vapor was used using Clevenger apparatus for 3 h. (Ahmadi, 2000). Moreover, essential oil yield was calculated by multiplying seed yield by essential oil percentage. 2.4. Statistical analysis The obtained data were subjected to analysis of variance (ANOVA) with SAS 8.1 software. The Bartlett test showed homogeneity in the variance of all traits in both years. Probabilities of significance (p ≤ 0.01 or 0.05) were used to test the significance among the main treatment effects and interactions. When an F-test indicated statistical significance, the protected least significant difference (LSD) was used to separate the means of the main effect, and the significant interaction effects were separated by slicing method. 3. Results and discussion 3.1. Analysis of variance Analysis of variance indicated that the main effects of the irrigation treatments (I), mycorrhizal treatments (M) and year were statistically significant on all parameters (Table 1). Moreover, the first order interactions (I × M, I × year and M × year) significantly influenced all studied traits, except for the following: (1) I × M on dry matter yield and essential oil percentage; (2) M × year on umbel number per plant and essential oil percentage and (3) I × year on umbel number per plant and essential oil percentage. The results

Please cite this article in press as: Gheisari Zardak, S., et al., Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of drought stress. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.09.004

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non-inoculated fennel Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

150

a a

a b

b

b

c

b a

Plant height(cm)

120

a a a

b b

b

b

a

b

b b

90

a

b a b

a b

a b c

b

b

a

c

a a b

60 30 0 Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Different irrigation treatments

2011

2012

Averaged over both years

Fig. 2. Interaction effect of irrigation treatments × mycorrhizal treatments on fennel height. Means followed by the same letter are not significantly different (P ≤ 0.05). Ww: well-watered; Mds: moderate drought stress; Sds: severe drought stress; VSds: very severe drought stress.

Table 2 Mean comparisons of irrigation and mycorrhizal treatment main effects on fennel traits. Treatments

Traits Plant height (cm)

Umbel number per plant

Grain number per umbel

Leaf P concentration (mg g−1 dry mass)

leaf soluble sugar content (mg g−1 fresh leaf weight)

Irrigation treatment

2011

2012

2011

2012

2011

2012

2011

2012

2011

2012

Well-watered (I1 ) Moderate drought stress (I2 ) Severe drought stress (I3 ) Very severe drought stress (I4 )

96a 89b 84b 83b

137a 136a 126b 119b

54b 64a 54b 62a

65b 69b 64b 72a

38b 46a 45a 43a

101b 111a 97b 62c

14.5b 24a 10c 13.6b

35.7a 34.7a 35.4a 30.9b

300d 405a 319c 323b

292ab 249c 285b 307a

Mycorrhizal treatment non-inoculated fennel Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

85ab 91a 88b

123c 134a 134b

37c 67b 71a

52b 71a 78a

62a 47b 45b

116a 63b 57c

16.6b 17.3a 17.1a

31.8c 35b 38a

302b 310b 464a

261c 289b 299a

Year average

88b

130a

58b

67a

51b

79a

17b

35a

359a

283b

Means within each column of each section followed by the same letter are not significantly different (p ≤ 0.05).

showed that the second order interaction (I × M × year) had significant effect on all traits, except for the effect of I × M × year on essential oil percentage (Table 1).

3.2. Mycorrhizal colonization The results indicated that drought stress (especially in I2 and I3 treatments) had a positive effect on AM colonization (data are not shown). The AM colonization under moderate and severe drought stress (I2 and I3 treatments) was higher than that under very severe drought stress (I4 treatment) and well-watered treatment (I1 ). Other researchers (Wu and Xia, 2006; Augé, 2001) have stated that root colonization more often increased than decreased under drought stress conditions. As Mardukhi et al. (2011) reported, under drought stress conditions more carbon is allocated to the roots, resulting in higher rate of root colonization and hence, symbiosis. The AM fungi used in this study were two morphologically distinguishable Glomus species. These organisms were able to grow in neutral to alkaline Mediterranean soils which are prone to water stress. Moreover, Irrespective of drought stress intensity, root colonization rate in non-inoculated plants was strongly lower than that compared with inoculated plants, in both years. This demonstrates the lack of any mycorrhizal activity in the soil. In general, the highest colonization was observed in roots inoculated with G. mosseae at each level of drought stress.

3.3. Plant height The results revealed that any reduction in water availability under drought stress treatments (I2 , I3 and I4 ) would reduce fennel height (Table 2). When averaged over both years and compared with well-watered treatment (I1 ), very severe drought stress (I4 ) decreased fennel height by 12 and 20 cm in AM and non-AM plants, respectively (Fig. 2). The plant height in G. intraradices plots was 3 and 5 cm higher than that in G. mosseae inoculated plots received moderate drought stress in the first and second year, respectively. By contrast, plant height in G. mosseae inoculated plots was 2 and 7 cm higher than that in G. intraradices inoculated plots treated with very severe drought stress in 2011 and 2012, respectively (Fig. 2). In well-watered plots, G. mosseae inoculated plants in the first year and G. intraradices inoculated plants in the second year were the highest plants, 101 and 144 cm height, respectively (Fig. 2). The reduction in plant height due to water shortage is in line with finding of Earl and Davis (2003). The decreased fennel height due to drought stress conditions can be related to the reduction in CO2 assimilation. On the other hand, positive effects of mycorrhizal inoculation are attributed to improved nutrients uptake (Bethlenfalvay et al., 1988) and greater water absorption by hyphae (Faber et al., 1991). Moreover, it has been reported that mycorrhizal symbiosis enhances the photosynthetic source of plants through increasing leaf area index (Gholamhoseini et al.,

Please cite this article in press as: Gheisari Zardak, S., et al., Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of drought stress. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.09.004

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Table 3 Mean comparisons for interaction of irrigation and mycorrhiza in the first and second year and compound analysis for umbel number per plant and grain number per umbel. Irrigation treatments

Mycorrhiza treatments

Umbel number per plant

Grain number per umbel

2011

2012

Compound analysis

2011

2012

Compound analysis

Well-watered (I1 )

non-inoculated fennel Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

37b 61a 64a

47c 66b 81a

42c 63b 73a

54a 33b 27b

138a 103b 95b

96a 68b 61b

Moderate drought stress (I2 )

non-inoculated fennel Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

30c 69b 92a

44c 77b 86a

37c 73b 89a

61a 30b 27b

139a 88b 106c

100a 59b 67b

Severe drought stress (I3 )

non-inoculated fennel Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

42b 47b 74a

51c 77a 63b

46b 62a 69a

45a 30b 53a

61b 81a 85a

53b 56a 69a

Very severe drought stress (I4 )

non-inoculated fennel Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

40c 65b 80a

65b 68b 82a

52c 67b 81a

27a 32a 39a

55a 61a 58a

41b 47a 49a

In each irrigation treatment and within each column, means followed by the same letter are not significantly different (p ≤ 0.05).

50 non-inoculated fennel

45

Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

Leaf P concentration (mg /g dry mass)

40

a

a b b

35

c

30

15

b a

c

a b

25 20

a

b

a a b

a b

c a

a

b

c

a

a b

b c

b

a

a

b

c

b c

a

b

10 5 0 Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Different irrigation treatments

2011

2012

Averaged over both years

Fig. 3. Interaction effect of irrigation treatments × mycorrhizal treatments on leaf P concentration. Means followed by the same letter are not significantly different (P ≤ 0.05). Ww: well-watered; Mds: moderate drought stress; Sds: severe drought stress; VSds: very severe drought stress.

2013), consequently plants with higher production capacities can produce higher plants.

3.4. Umbel number per plant (UNPP) and grain number per umbel (GNPN) UNPP and GNPN are considered as the most important morphological traits in fennel plant, and their reduction due to drought stress could reduce fennel yield. A comparison between the combined treatments indicated that under well-watered treatment (I1 ) and under drought stress conditions (I2 and I4 treatments), G. mosseae inoculation significantly increased UNPP compared with G. intraradices or control treatment (Table 3). It can be concluded that mycorrhizal symbiosis accelerates flowering (data not shown) and increases UNPP by improving plant nourishment and increasing plant growth. The results are in line with finding of Kapoor et al. (2004) who related the higher UNPP in fennel plant to more mineral nutrient availability specially P and more biological yield in mycorrhizal inoculated plants than non-inoculated plants. However, G. mosseae responded more effectively, as it resulted in a significant increase in UNPP compared with non-inoculated plants. It has been reported that G. mosseae is more able to penetrate into the host roots compared with other species (Martin et al., 2012). In addition, in both years, the highest GNPN was observed in moderate drought stress treatment (Table 2). In free AM plants, an increase in drought stress intensity (from I1 to I4 treatment) decreased GNPN by 55 unites. In AM inoculated plants, this reduction in water availability decreased GNPN by 17 unites. It appears

that the higher soil root density in AM inoculated plants is effective in reducing the sensitivity of the inoculated fennels to water deficiency. Studies on non-AM plants root distribution showed that root growth occurs in a series of stages associated with the plant developmental stages (Boomsma and Vyn, 2008). By contrast, the AM plants were observed to have deep and rapid root expansion (Shahhosseini et al., 2012), a characteristic that enable them to successfully extract water.

3.5. Leaf P concentration and leaf soluble sugar content Osmotic adjustment in plants specifically involves the active accumulation of various ions, amino acids, and sugars (Chaves et al., 2003). It is generally necessary for the maintenance of turgor pressure, cellular expansion and growth as well as stomatal opening, photosynthesis, and water influx during water stress (Morgan, 1984; Ruiz-Lozano, 2003). Phosphorus concentration in leaves may affect stomatal response to environmental stresses, by affecting the energetics involved in guard cell osmotic parameters or wall stiffening governing stomatal movements (Augé, 2001). In both experimental years and in non-AM plants, the irrigation treatments showed a significant effect on leaf P concentration leaf P concentration decreased with increasing drought stress intensity (Fig. 3). By contrast, the leaf P concentration in inoculated plants was less affected by drought stress. The results showed that very severe drought stress (I4 treatment) decreased non-AM fennel leaf P concentration by 27 and 15% (compared to well-watered treatment) in 2011 and 2012, respectively. On the contrary, I4 treatment did not

Please cite this article in press as: Gheisari Zardak, S., et al., Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of drought stress. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.09.004

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non-inoculated fennel Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae a

500

leaf soluble sugar content (mg/g fresh leaf weight)

450 400

a a

a

a

350

a

300

b

a b

b

b

a

b b

b

250

a

a

b

c

c

b

b

a

b

b

b

a

a

b

b

b

b c

c

c

c

200 150 100 50 0 Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Different irrigation treatments

2011

2012

Averaged over both years

Fig. 4. Interaction effect of irrigation treatments × mycorrhizal treatments on leaf soluble sugar content. Means followed by the same letter are not significantly different (P ≤ 0.05). Ww: well-watered; Mds: moderate drought stress; Sds: severe drought stress; VSds: very severe drought stress.

Table 4 Mean comparisons of irrigation and mycorrhizal treatment main effects on fennel traits. Traits Treatments

Dry matter yield (kg ha−1 )

Seed yield (kg ha−1 )

Essential oil percentage

Irrigation treatment

2011

2012

2011

2012

2011

2012

2011

2012

Well-watered (I1 ) Moderate drought stress (I2 ) Severe drought stress (I3 ) Very severe drought stress (I4 )

10116b 12013a 10837b 10620b

13322b 14585a 12476bc 11658c

1133b 1338a 1129b 1191b

3732b 4441a 3538b 2326c

2.73b 3.05a 2.88b 3.06a

3.10c 3.84a 3.42b 3.12c

31.2b 40a 32.4b 33.8ab

114.1c 177.9a 125.4b 73.6d

Mycorrhizal treatment non-inoculated fennel Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

10927a 11130a 10631a

12135b 13725a 13170a

1198a 1177a 1218a

3171b 3733a 3624a

2.85a 2.96a 2.92a

3.05b 3.51a 3.51a

33.8a 34.3a 35.7a

101c 137.8a 129.5b

Year average

10869b

13010a

1198b

3509a

2.91b

3.35a

34.6b

122.7a

Essential oil yield (kg ha−1 )

Means within each column of each section followed by the same letter are not significantly different (p ≤ 0.05).

significantly affect leaf P concentration of the inoculated fennels (Fig. 3). In other words, it is likely that mycorrhizal symbiosis can increase root proliferation, P uptake per unit root weight and/or the translocation of P to the aboveground portion of the plant even under very severe drought stress conditions. Higher leaf P concentration in AM plants compared with non-AM plants after drought stress, suggests maintenance of greater photosynthetic capacity (Subramanian and Charest, 1997; Augé, 2001), which is attributed to greater drought resistance of AM plants. Davies et al. (2002) declared that increase in leaf P concentration in AM plants is associated with improved water relation in pepper. Although AM fungi can promote P uptake, and P uptake is generally reduced under drought conditions (Table 2), it is not very clear how increased P uptake could improve drought resistance. However, it has also been reported that the effect of AM fungi on drought resistance may be independent of P uptake (Ruiz-Lozano et al., 1995). In general, the involvement of P in drought resistance of AM plants is controversial. In addition, the results showed that G. mosseae inoculated plants accumulated more P in their leaves compared with G. intraradices inoculated plants or non-inoculated plants grown under well-watered treatment or sever and very severe drought stress treatments, in both years (Fig. 3). Results suggest that this species is the best adapted, or the most aggressive colonizer, especially under severe and very severe drought stress conditions. Mardukhi et al. (2011) declared that relationship between AM fungi and the host plant is non-specific, however, under stress condi-

tions; there may be combinations of AM-host plant, which are more efficient. Leaf osmotic adjustment based on the organic solutes, including soluble sugar, in mycorrhizal plants has been reported as a resistance mechanism to drought stress (Wu and Xia, 2006). As expected, for AM plants in both years an increase in drought stress intensity led to increase in leaf soluble sugar concentration (Fig. 4). The positive effect of mycorrhizal symbiosis on leaf soluble sugar content was higher with high intensity of drought stress compared with low intensity of drought stress. Averaged over both years, in the moderate (I2 treatment) and very severe (I4 treatment) drought stress, mycorrhizal symbiosis increased leaf soluble sugar by 22 and 51% (compared with well-watered treatment), respectively. Greater sugar concentration in leaves of AM inoculated plants may have resulted from not only potentially improved leaf area, but also reduced chlorophyll photooxidation (Balestrini and Lanfranco, 2006), higher leaf water potential values (Boomsma and Vyn, 2008), and higher photosynthetic rates (Smith and Read, 1997). Furthermore, the results showed that under severe and very severe drought stress conditions, G. mosseae inoculation led to a significant increase in leaf soluble sugar content compared with the G. intraradices inoculated fennels (Fig. 4). By contrast, G. intraradices inoculated plants produced higher leaf soluble sugar in comparison with G. mosseae inoculated plants grown under moderate drought stress conditions (Fig. 4). Difference between mycorrhizal species with regard to leaf osmotic adjustment might be due to the differences in their origin. The G. mosseae species was selected from arid regions in Iran, which

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non-inoculated fennel

14000

Dry matter yield (kg/ha)

12000

a

Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

16000

a a a a

10000

a

b

a a a

a a

a a a a

b

b c

a

b

a a

b

a a

a

a a

a

bb

a a a

b

8000 6000 4000 2000 0 Ww (I1)

Mds (I2) Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2) Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2) Sds (I3)

VSds (I4)

Different irrigation treatments

2011

2012

Averaged over both years

Fig. 5. Interaction effect of irrigation treatments × mycorrhizal treatments on dry matter yield. Means followed by the same letter are not significantly different (P ≤ 0.05). Ww: well-watered; Mds: moderate drought stress; Sds: severe drought stress; VSds: very severe drought stress.

enhanced its ability to confer drought resistance of the host plants. Conversely, G. intraradices species was originated from the humid area of Iran. In addition, differences between mycorrhizal species linked to leaf soluble sugar may have resulted from G. intraradices acting as a stronger sink for available sugar (Schellenbaum et al., 1998; Amerian et al., 2001) or with more extensive colonization by G. mosseae than G. intraradices, especially under severe drought stress conditions (data not shown). Lower leaf soluble sugar content obtained from AM inoculated fennels compared with non-AM fennels under well-watered treatment (Fig. 4). This might be due to this fact that maintaining AM associations in the roots has a cost in translocation of sugar to the root system. Varying by plant and fungal species, plant age, and AM developmental stage, AM can consume between 2 and 20% of daily host assimilate production (Graham, 2000; Bryla and Eissenstat, 2005). As mentioned earlier, the results showed that leaf soluble sugar was higher in non-inoculated plants than those of inoculated plants under well-watered treatment (Fig. 4). These results are in contrast with other researchers (Schellenbaum et al., 1998; Augé, 2001). Schellenbaum et al. (1998) stated that the AM fungi are stronger competitor for root-allocated carbon when plants endue drought stress conditions. Two factors explain these results. First with a high drought stress intensity fennel plants provide little benefit to the AM fungi and second, the ability of fennel plants to use AM fungi efficiently decreases under well-watered conditions because

of reduction in colonization rate. No one has reported the influence of mycorrhizal symbiosis on the leaf soluble sugar content of fennel under stress and non-stress conditions.

3.6. Dry matter yield and seed yield According to the results for both inoculated and non-inoculated plants increase in drought stress intensity, especially under very severe drought stress (I4 treatment) in the second year, could led to significant reduction fennel dry matter and seed yield (Table 4). Averaged over both years, I4 irrigation treatment reduced AM inoculated fennel dry matter and seed yield by 3 and 20% and nonAM fennel dry matter and seed yield by 9 and 27%, respectively (Figs. 5 and 6). This difference might be due to stronger root system in AM inoculated plants and/or the higher transpiration and photosynthetic rates (Druge and Schonbeck, 1992), higher stomatal conductance and lower leaf temperature in these plants (Wu and Xia, 2006) compared with non-inoculated plants, especially under drought stress conditions. In other words, in AM inoculated plants, mycorrhizal symbiosis inhibits negative effects of water shortage on fennel dry matter and seed yield. According to the results of both years, the effects of AM fungi on alleviation of drought stress became greater with increasing drought stress intensity. Additionally, the results showed that inoculation of fennel plants with AM in drought stress conditions results in similar or

non-inoculated fennel

a

Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

5000 4500

b a

4000

c

Seed yield (kg/ha)

3500

b c

a

a

3000

a

b

b

c

c

2500 c

2000 1500 1000

a

b

a

a a a

a

b b

c

b

b

b

b a

c

a a

b

a c

a

500 0 Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2) Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Different irrigation treatments

2011

2012

Averaged over both years

Fig. 6. Interaction effect of irrigation treatments × mycorrhizal treatments on seed yield. Means followed by the same letter are not significantly different (P ≤ 0.05). Ww: well-watered; Mds: moderate drought stress; Sds: severe drought stress; VSds: very severe drought stress.

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non-inoculated fennel

220 200

Inoculated fennel with G. intraradices Inoculated fennel with G. mosseae

a b

180

Essential oil yield (kg/ha)

160 140

a a

c

a

a

b

100

b a

80 60 40

a

b

120

c a a a

a a a

Ww (I1)

Mds (I2)

ba a

a

a a

a

b b b

a ab a

b b

a a

20 0 Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Ww (I1)

Mds (I2)

Sds (I3)

VSds (I4)

Different irrigation treatments

2011

2012

Averaged over both years

Fig. 7. Interaction effect of irrigation treatments × mycorrhizal treatments on essential oil yield. Means followed by the same letter are not significantly different (P ≤ 0.05). Ww: well-watered; Mds: moderate drought stress; Sds: severe drought stress; VSds: very severe drought stress.

even higher seed yield production with non-AM fennel, which received full irrigation (Fig. 6). These results demonstrate that if optimum symbiosis of AM fungi is available, fennel will have a considerable capacity to produce high seed yields, even at low water conditions. Findings of Sajjadnia et al. (2013) on Fennel (Foeniculum vulgare Mill.) have also been supported the results of this study. It has been shown that associations between roots and AM fungi enhance the amount of P taken up by plant roots under drought conditions (Ruiz-Lozano et al., 1995). As a result of increased P uptake, inoculated plants with mycorrhizae frequently produce higher dry matter and seed yield than those without AM fungi. Moreover, improved P uptake due to presence of AM fungi during periods of water shortage has been postulated as a primary mechanism for the enhanced drought tolerance of the host plants (Bethlenfalvay et al., 1988). Greater plant P content has been suggested to directly improve drought tolerance through improved stomatal abscisic acid sensitivity (Boomsma and Vyn, 2008), greater stomatal conductance (Porcel and RuizLozano, 2004), and increased transpiration (Wu and Xia, 2006). There are two important mechanisms by which P availability and absorption can be enhanced through the inoculation of mycorrhizal fungi: firstly, by enhancing the P uptake that is facilitated by the extensive hyphae of the fungus, allowing them to explore more soil volume than can the non-AM plants (Evelin et al., 2009) and secondly, by increasing the microbial biomass in the mycorrhizal soil. Gholamhoseini et al. (2013) reported that microbial biomass increases when AM fungi are added into the soil, this leads to increase in soil CO2 content, which in turn forms H2 CO3 in the soil solution. This weak acid can dissolve primary phosphoruscontaining minerals, thereby increasing P availability. The positive effect of G. mosseae inoculation on fennel seed yield, especially in the second year, was highest in I2 treatment (Fig. 6). The difference between G. mosseae inoculated plants with G. intraradices inoculated plants reached 225 and 272 kg seed ha−1 in the first and second year under moderate drought stress treatment, respectively (Fig. 6). The mycorrhiza symbiosis and importantly the superior of G. mosseae to uptake more water and nutrients most likely explain the results. In fact, G. mosseae inoculated plants use water more efficiently than G. intraradices inoculated plants to produce seed during increased drought stress (especially under moderate and very severe drought stress treatments). In other words, the results showed that G. mosseae was a more effective fungal symbiont for increasing the drought tolerance of fennel, both in terms of growth under drought conditions and in terms of yield productivity. Rapid growth of G. mosseae hyphae suggested by Jansa

et al. (2008) might be expected to result in improvement in fennel growth relative to hyphae of slower growing G. intraradices species.

3.7. Essential oil percentage and essential oil yield Seed essential oil content increased by 15% in 2012 compared with 2011 (Table 4). This is due to better establishment of fennel plants in the second year compared with the first year. In both years, well-watered treatment (I1 ) showed the lowest seed essential oil content, being 11% lower than for very severe drought stress treatment (which had a maximum essential oil percentage in 2011) and 19% lower than for moderate drought stress treatment (which had a maximum essential oil percentage in 2012) (Table 4). Khalid (2006) reported that secondary products of medicinal plants can be altered by environmental factors and drought stress is a major factor affecting the synthesis of natural products. The results showed that drought stress increases the fennel essential oil percentage because under stress condition, more secondary metabolites are produced in plants to reduce oxidization in the cells. In both years, there was significant difference between inoculated and non-inoculated fennel plants with regard to seed essential oil content so that the AM inoculated fennel plants produced 2.94 and 3.51% (in the first and second year, respectively) and non-AM inoculated fennel plants produced 2.85 and 3.05% seed essential oil in 2011 and 2012, respectively (Table 4). Unfortunately, there is no comprehensive information indicating how mycorrhizae affect seed essential oil percentages in medicinal plants. Increase in essential oil percentage due to application of mycorrhizae might be due to increase in P availability in plants because of mycorrhizal association. Additionally, essential oil in medicinal plants such as fennel is based on the terpenoid compounds (Omidbeigy, 2000) and their composer units need the different elements especially P. Therefore, improved P uptake on account of AM fungi has been postulated as a primary mechanism for the enhanced seed essential oil content in fennel. Essential oil yield in medicinal plants reflects not only seed quality but also production quantity. As shown in Fig. 7, the G. mosseae inoculated fennel plants grown under moderate drought stress showed the highest essential oil yield in both years of the experiment (42 and 206 kg ha−1 in 2011 and 2012, respectively), whereas the non-inoculated fennel plants grown under severe drought stress treatment in the first year (29 kg ha−1 ) and under very severe drought stress treatment in the second year (63 kg ha−1 ) showed the lowest essential oil yield. In 2011, there was no considerable difference between mycorrhizal species, however in 2012, essen-

Please cite this article in press as: Gheisari Zardak, S., et al., Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of drought stress. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.09.004

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tial oil yield was significantly higher in G. mosseae inoculated fennel plants grown under different irrigation treatments compared with other mycorrhizal treatments (Fig. 7). From both years, essential oil yield in G. mosseae inoculated fennel plants were 3 and 29% in plants grown under well-watered, 7 and 44% under moderate drought stress, 15 and 5 under severe drought stress and finally 7 and 40% under very severe drought stress conditions higher than essential oil yields for fennel inoculated with G. intraradices and non-inoculated fennel, respectively. In fact, the G. mosseae inoculated plants supplied the acceptable amount of essential oil yield for most irrigation treatments so that this species was found to be more effective fungal symbiont for increasing fennel essential oil yield. The data presented here show that different AM fungi species even within the same genus have distinct effects on medicinal plant growth and yield. The symbiotic behavior of an AM fungal species depends on its ability to colonize the roots of a host plant rapidly and extensively. The first factor influencing fungal infectivity is the dormancy of spores (Walker et al., 1995) which occurs in some species of AM fungi and can negatively influence infection, delaying the formation of germinative hyphae and appressoria (Tommerup, 1983). It is clear that different mycorrhizal species, because of different patterns of spore dormancy and different penetration ability to host roots, have different root colonization capacity and also have different influence on plant growth. The results demonstrated that G. mosseae can improve fennel quantity and quality yield and drought resistance to a greater level than G. intraradices. These improvements are attributed to greater colonization of fennels by G. mosseae compared with G. intraradices. A higher colonization rate in G. mosseae inoculated fennel plants compared with G. intraradices inoculated plants grown under different irrigation treatments (especially drought stress conditions), might be due to a greater adaptation of this species to its native soil and climate, in the sense of benefiting its hosts best, given these soil-climate conditions. 4. Conclusion In conclusion, the results reveal that AM inoculation significantly affect quantitative and qualitative yield in fennel plants. Since fennel is a mycorrhizal dependent plant, the inoculation with AM fungi is an excellent strategy to enhance the benefits of the symbiosis. Furthermore, selection of AM fungi for introducing into dry environments to address specific problem situations is a promising but usually neglected strategy. The results also clearly illustrate that G. mosseae is more efficient under drought stress so that supports fennel plants. The application of these microorganisms could be critical in cultivation of medicinal plants under arid and semi-arid regions, where water is the most important factor in determining plant growth and yield. References Ahmadi, L., 2000. Identifying the compositions of the essential oil from cumin. Iranian Journal of Medicinal and Aromatic Plant 6, 97–113. Amerian, M.R., Stewart, W.S., Griffiths, H., 2001. Effect of two species of arbuscular mycorrhizal fungi on growth, assimilation and leaf water relations in maize (Zea mays). Aspects of Applied Biology. 63, 73–76. Augé, R.M., 2001. Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza 11, 3–42. Azcón, R., Gómez, M., Tobar, R., 1996. Physiological and nutritional responses by Lactuca sativa L: to nitrogen sources and mycorrhizal fungi under drought conditions. Biology and Fertility Soils 22, 156–161. Balestrini, R., Lanfranco, L., 2006. Fungal and plant gene expression in arbuscular mycorrhizal symbiosis. Mycor 16, 509–524. Bethlenfalvay, G.J., Brown, M.S., Ames, R.N., Thomas, R.E., 1988. Effects of drought on host and endophyte development in mycorrhizal soybeans in relation to water use and phosphate uptake. Physiologia Plantarum 72, 565–571. Bethlenfalvay, G.J., Brown, M.S., Franson, R.L., Mihara, K.L., 1989. The Glycine-Glomus-Bradyrhizobium symbiosis. IX. Nutritional, morphological

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Please cite this article in press as: Gheisari Zardak, S., et al., Responses of field grown fennel (Foeniculum vulgare Mill.) to different mycorrhiza species under varying intensities of drought stress. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.09.004