Effect of different ferric fertilizers on planting Morchella conica fruiting yields and analyses of the microflora and bioactivities of its grown soil

The aims of this study were to determine whether six ferric fertilizers influence the development of fruit bodies of Morchella conica identified by analysis of the internal transcribed spacer (ITS), its grown soil microflora and enzymatic activities. We successfully cultivated one kind of Morchella in the wheat planting field, its partial ITS rDNA in the present study was identified as M. conica by GenBank and Morchella MLST database as a reference. Six ferric fertilizers were applied to the planting area. Four soil enzymatic activities (including soil urease, polyphenol oxidase, invertase, and protease) as well as soil microflora showed significant differences in the development stages of M. conica especially during the primordium differentiation stage. Results suggest that ferric fertilizer not only influenced the Morchella grown soil enzymatic activities and microorganisms, but also increased the Morchella yields while such effects differ from the species of the ferric fertilizer, especially groups with ammonium ferric sulfate [NH4Fe(SO4)2.12H2O], Fe·EDDHA and Tongfeng nutrients. So, this work highlights the importance of further attempts to resolve important aspects of the morel commercial cultivation regarding optimizing nutrition plan and figuring out the relationships between genotypes and their fertilities of fruiting body.


INTRODUCTION
In the world's 1.5 million estimated fungi species (Hawksworth, 2004), few genera are as synonymous with epicurean cuisine as true morels (Du et al., 2012b), highly prized for their medicinal and nutritional qualities (Kanwal and Reddy, 2012).Fruiting bodies of morel fungi (Morchella spp., phylum Ascomycota) are highly restricted to tem-perate regions of the Northern Hemisphere in a few weeks each spring (O'Donnell et al., 2011).Due to the increasing popularity and commercial demand, wild morels are harvested commercially and exported extensively from China, India,Turkey, Mexico, and the United States (Pilz et al., 2007;Taskin et al., 2010).*Corresponding author.E-mail: Lzf0028@163.com.
The first successfully growing morel fruit body under controlled condition was carried out by cultivating in a specialized indoor facility using patented technology within the US (Ower et al., 1986), included outdoors in Yunnan Province of China (Zhao et al., 2009).
Morels' very short harvesting season and high price have encouraged researchers to study the biology and ecology of the species (Goldway et al., 2000).Many researchers have attempted to raise morels in controlled conditions; nevertheless, ascoscarps reaching the market are mostly collected from the wild (Ower et al., 1986;Goldway et al., 2000;Masaphy, 2005;Guler and Ozkaya, 2009;Masaphy, 2010).Their modes of reproduction, biology, nutritional sources, life cycle, are unusual and complex (David et al., 2007).Recently, a remarkable finding is that the majority of the Esculenta clade appeared to be adapted to mixed temperate deciduous hardwood forests with the Elata clade's preference to an evergreen coniferous biome (Du et al., 2012a;Donoghue, 2008).By all odds, it is important showing the morels growing conditions.
Previous reports are almost focused on their mycelial fermention or officinal functions, little on their cropping stages investigation.The mating systems of Morchella species are unknown (Buscot, 1993), and whether heterokaryosis (mating of two compatible strains) followed by meiosis occurs in natural populations of morels is also unknown (Volk andLeonard, 1989, 1990).Attention has focused on antioxidant activities of produced compounds, content of phenolics and flavonoids, autofluorescence and various metals (Gursoy et al., 2009;Kalač 2010).Many studies have demonstrated that minerals and heavy metals play an important role in the metabolic processes, during the growth and development of mushrooms in appreciable concentration (Claudia et al., 2010) and wild-growing mushrooms can accumulate great concentrations of toxic metallic elements and metalloids such as mercury, cadmium, lead, copper or arsenic and zinc (Vetter, 2004;Nevcihan et al., 2009;Zhu Fangkun et al., 2011).Reports pointed out that the accumulation of metals in fungi found to be affected by element environment such as organic matter amount, temperature, humidity, pH, metal concentrations in soil and fungal factors such as species of microfungi, morphological part of fruiting body, development stages and age of mycelium, biochemical composition, and interval between the fructifications affect metal accumulation in macrofungi (Mendil et al., 2005;Nevcihan et al., 2009).
It has also been shown that Fe was abundant in seven Morchella species, follow by calcium and magnesium, respectively (Nevcihan et al., 2009).Iron can stimulate lipid peroxidation by the fenton reaction, and also accelerates peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals that can abstract hydrogen and perpetuate the chain reaction of lipid peroxidation (Halliwell, 1991).As the most effective pro-oxidants, ferrous ions are commonly found in food systems (Yamaguchi et al., 1998).
Based on our previous study, we successfully cultivated Morchella in the wheat planting field.Up to now, the formation of fruit-bodies of morchella is still mysterious and their commercial production is not mature.Commercially cultivation of morel has also been confronting with series of difficulties.Many triggers for fruiting appear to differ by species to some extent.We still do not know which vital factor determines that the transformation from sclerotium of Morchella to primordium of fruit bodies, such as soil microorgansiam or microelements or other compounds, even the differences existing in genomices between fertile and infertile morchella strains.
In the present paper, our aims are to identify the morels using internal transcribed spacer (ITS) data with Morchella MLST as a reference and the effect of the different ferrous treatments on soil characteristics and the morel yields, in order to determine the possibility of optimal morels growing conditions.

Strains and chemical agents
pMD18-T Vector Ligation Kit, DL2000 DNA Marker was purchased from TaKaRa Technology (Dalian) Co., Ltd.DNA Gel Extract Kit was purchased from MBI Fermentas.Morchella conica strain Mc-5 (commercially cultivated for five years), Escherichia coli DH5α and BL21 (DE3) strains were preserved in author's Laboratory.All other reagents except the noted agents used in present study were of analytical grade.

Molecular identification of Morchella strain and its sclerotia observation by scanning electron microscopy (SEM)
Total genomic DNA of M. conica was followed by a hexadecyl -trimethyl-ammonium bromide (CTAB) protocol (O'Donnell et al., 1997;Taşkin et al., 2010) when the mycelium was freeze-dried before the CTAB extraction step.The internal transcribed spacer (ITS) rDNA sequence was amplified using the total DNA as template while the primer pair of ITS1 and ITS4 was used as the primer pair, upstream primer (ITS1: 5'-TCCCTTACTCTTCTAACCTCTCCTCT-3') and downstream primer (ITS 4: 5'-TCCTCCC TCTTATTCTATATCTC-3'). Polymerase chain reaction (PCR) products were purified with the DNA Extraction Kit (MBI Ferments).The rDNA fragment was T-A cloned into the pMD18-T vector (Takara Shuzo Co., Ltd., Dalian, China) and propagated in E. coli (strain DH5α).
The positive clone was screened by Clone-PCR method (Gussow and Clackson, 1989;Sathe et al., 1991).The ITS sequences of the isolates was determined by Shanghai Jikang Biotech Company (Shanghai, China), then used as templates to search for homologous sequences in GenBank using the Basic Allignment Search Tool (BLAST) and Morchella MLST database as a reference (Robert et al., 2011).The obtained sequences were compared one to another by the computer-assisted method of Higgins and Sharp (1988).The phylogenetic relationships within the Morchellaceae using a aligned nucleotide positions in Paup via one thousand Maximum parsimony bootstrap (BS).
Experimental strain of M. conica Mc5 was grown in potato dextrose agar (PDA) and its sclerotia was harvested and fixed for 2 h at 4°C by using 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer.Secondary fixation was performed in 1% osmium tetroxide in dH2O after the samples were washed three times with 0.1 M cacodylate buffer (pH 7.4), and were then treated with 1% (w/v) osmium tetroxide, washed with 5% (w/v) sucrose in cacodylate buffer, and subsequently dehydrated in a graded ethanol series (from 30 to 100%).The samples were examined on a JEOL JSM-7500F Scanning Electron Microscopy after the works for critical point drying and gold coating were successfully carried out and the images were recorded.

Cultivation Experimental groups sprayed with ferric fertilizers
First fruit bodies pieces collected for pure M. conica culture isolation, sprayed with 70 % ethanol, were grown well in the PDA (potato 20 g/L, glucose 2g/L, agar 2g/L) test-tubes, then sub-culture of isolates were removed in another new sterilized medium which consists of wheat 70%, vermiculite 5%, pure soil 15% and chaff 10%, natural pH value and cultured under 15-18°C since mid-October for over 15 days, when morel sclerotia were formed well.
We choose Longxing Town (located in Chongzhou, Sichuan Province, China) where hardwood trees grow naturally and soil is sandy loam or humus without much clay.The climate is subtropical with annual precipitation and average temperature of 1012 mm and 17.8°C.Each section was 10×0.6 m 2 .Each treatment was established with three replicates.Seeding of Morchella spawn was carried out during early November and about 1.5 bottle (750 mL/bottle) spawn were used per square meter (m 2 ) when the planting field tamped and leveled up firmly.About twenty days later after seeding in December, six experimental groups, including Iron II ethylenediamine di(o-hydroxyphenylacetic) acid (abbreviated as Fe•EDDHA, provided by Sichuan Tongfeng Science and Technology Co. Ltd, Chengdu, China), ferrous sulfate (FeSO4.7H2O),ferric sulfate [Fe2(SO4)3.9H2O],ammonium ferric sulfate [NH4Fe(SO4)2.12H2O],citrate ferric, Tongfeng nutrition agents (abbreviated as nutrients, also provided by Sichuan Tongfeng Science and Technology Co. Ltd, Chengdu, China) with six trace elements (including with Fe, Zn, Mn, Mo, B, Cu, and its ferric content was about 2%), were separately sprayed onto the soil sampling districts grown with M. conica, and all the nutrition concentration of different ferric fertilizers was 100 mg/m 2 when Morchella mycelium grown well in the field before sclerotia formation, while the same volume of distilled water was sprayed as the control.Primordia formation started as temperatures increase above 10°C (a range between 6-15°C), lasting for one month since late January.The fruiting bodies appear approximately in the early February, obviously influenced by the soil temperature, and the fruiting season is no better than 20 days, compared with wild morels in ten days in other reports (Mihail et al., 2007;Wurtz et al., 2005).

Enumeration of major soil microbial population groups
Soil samples were randomly collected aseptically in ultra-violet (UV) sterilized plastic bags from each groups to a depth of 10~30 cm during sclerotia formation, primordia formation and fruit-body growth separately in late November, late January and early February.The soil samples were kept on ice during transport to the laboratory and stored at 4°C before microbial enumeration.The enumeration of the soil microflora was done by the dilution plate method (Nair and Subba-Rao, 1977).Soil sampling (10 g) was done from three replicated sites and emulsified in 90 mL sterilized water, aseptically.A serial decimal dilution was made from this suspension up to 1:10 -10 . 1 mL of each dilution was used to inoculate plates in triplicate containing specific growth media for different microorganism.The total colony forming units (cfu) of bacteria, fungi and actinomycetes were recorded on Gauze No. 1 agar (for actinomycetes, Ye et al., 1983), Martin's rose bengal streptomycin agar agar (for fungi, Martin, 1950) and Jensen's agar (for bacteria, Jensen, 1951) media, respectively.There were nine plates for each sample, and the plates were incubated at 28°C for 2-4 days for bacteria, 5-8 days for fungi, and 11-14 days for actinomycetes; after, microbial population was calculated and expressed as 10 n cfu/g air dried soil, where 10 n was dilution factor.

Determination of enzymatic activity for Morchella grown soil
Soil samples were collected on the same time as the enumeration of major soil microbial population.The samples for the determination of enzyme activities were sieved (1 mm) after being air-dried at room temperature for 7 days and stored at 4°C prior to analysis.The studied soil enzymes were invertase, urease, polyphenol oxidases and proteases.Invertase activity in soil was assayed by measuring the release of reducing sugar (Papa et al., 2010).Soil urease activity was determined by the method of Tabatabai and Bremner (1972) and protease activity by the method of Ladd and Butler (1972) using gelatin as the substrate.Polyphenol oxidase activity was measured by absorbance at 420 nm using the method described by Kar and Mishra (1976) and pyrogallol used as the substrate.

Statistical analysis
All the experimental results were means±SD and statistical significances of differences among treatments were determined by using the Statistical Package for the Social Sciences (SPSS) (version 13), and followed by comparisons at significance level of 0.05 (P<0.05).

Morchella strain identification and its sclerotia observation by SEM
The edible mushroom Morchella esculenta Pers.forms sclerotia, whose importance in its life cycle was demonstrated by Ower et al. (1986).Amir at al. (1995) compared nine inhibitors affecting translocation and sclerotia formation in M. esculenta and they found that the use of nikkomycin inhibited sclerotia formation, without affecting translocation to the sclerotia.Therefore, it is very important to qualify the formation of sclerotia for the experimental strain.Results of scanning electron microscopy (SEM) had shown that Morchella sclerotia were formed by tube-shape mycelium and looked like granules (Figure 1).From the photos shown in Figure 1, we found that the sclerotia of M. conica were about 15±3.24 μm in diameter, and its mycelium about 10.25±2.13μm in diameter.Some mycelia were observed to create plasma bridges and such results were reported by others (Guler and Ozkaya, 2008;Arkan 1992).Volk and Leonard (1990) reported that one area of neglect is the cytology of various development stages of the Morchella life cycle: the vegetative hyphae, sclerotia and fruiting bodies.1A, mycelium of M. conica observed by SEM with the mycelium shaped as a kind of tube coated with small particles.Barrel-shaped cell in sclerotia.Bar= 1 μm. Figure 1B, sclerotia of M. conica were about 15±3.24 μm in diameter, and its mycelium about 10.25±2.13μm in diameter.Bar= 1 μm. Figure 1C, round, closely packed basal sclerotia.investigated the effects of various carbon sources for M. conica sclerotium formations under in vitro conditions and found sclerotia cells were generally very thick and spherical.It was thought that the angle of hypha growth was something collected with sclerotia in culture medium, but this needs further confirmation.
Former species-level molecular systematic markers employed within this genus were limited to isozymes (Gessner et al., 1987;Royse and May, 1990;Wipf et al., 1996), restriction fragment polymorphisms of the LSU rDNA (Bunyard et al., 1994(Bunyard et al., , 1995)), ITS region of the nuclear rDNA (Buscot et al., 1996), and phylogenetic analyses of the ITS rDNA sequence data (Wipf et al., 1999;Kellner et al., 2005).Using multilocus DNA sequence data and phylogenetic species recognition (Dettman et al., 2003;Taylor et al., 2000), researcher investigated DNA sequences from marker loci，including ITS rDNA, and partial sequences of LSU rDNA, RPB1, RPB2, and EF-1 alpha to study morels species limits and to assess evolutionary relationships (O'Donnell et al., 2011;Tas-kın et al., 2010Tas-kın et al., , 2012;;Du et al., 2012a).Du's research also found that ITS sequence data has limited utility in differentiating species within the species-rich Elata sub-clade in the identified three lineages within Morchella (Du et al. 2012b) and most Morchella species appear to exhibit continental endemism and provincialism (O'Donnell et al., 2011).Because two-thirds of the Morchella ITS rDNA sequences identified to species in GenBank are misidentified and the ITS rDNA partition still was the most length variable (Du et al., 2012a), the Morchella strain in the present study was identified by Morchella MLST database in preference to NCBI GenBank numbers.Therefore, the studied strain was authenticated by analyzing its partial ITS rDNA sequences (750bp, Wipf et al., 1996) and the homologies analyses with Morchella MLST database with GenBank as a reference.Results are shown in (Figure 2).
ITS rDNA sequences give some information to analyses the commercial cultivation strain.Conducting these sequences, either through the species identification or possibly establish species DNA barcoding, we can better contribute to more suitable breeding in future.From above results, we found a very interesting phenomenon, that the present study strain of M. conica are just the same as another strain sampled from North Israel when their ITS rDNA sequences were compared, which suggested disjunct distribution during their evolutionary relationships.

Enumeration of major microbial populations from grown soil for M. conica
Up to now, we know little about the changes of the microorganisms from the cultured morel soils, especially treated with microelements such as ferric, zinc, among others.Microorganisms require iron for growth.Under iron-limited conditions, low molecular weight compounds called siderophores are synthesised by microbes (Varma and Chincholkar, 2007).Siderophores chelate ferric ions and transport it to the cell via receptor-mediated way.As iron is an indispensable growth factor, microbes producing siderophores can capture it from the others.Iron-deprivation causes decreasing metabolic activity and stop cell growth.Pseudomonas species synthesise numerous iron-chelators, e.g.pyoverdin and pyochelin (Cox et al., 1981;Cox and Adams, 1985).These compounds play an important role in restricting the growth of fungi (Matthijs et al., 2007).In this study, the numbers of culturable microbial population in soil at different developing stages were measured and results (Table 1) showed that experimental groups were higher than the control in number of bacteria, fungi and actinomycetes (P<0.05).
In detail, groups treated with ferric citrate and Fe•EDDHA during spore-formation stage show no significant differences as compared to the control group in bacteria number, but the number of fungi significantly increased and actinomycetes' number were significantly decreased.During primordium differentiation stage, experimental groups except the groups treated with Fe 2 (SO4) 3 •9H 2 O, were all significantly increased in the bacteria, fungi and actino mycetes' numbers, but the group treated with ammonium ferric sulfate (NH 4 Fe(SO 4 ) 2 .12H 2 O) during spore formation were higher than others (included the control group) in the microorganism number, and such tendency was found in soil microflora number during the primordium differentiation stage.It is interesting that numbers of soil microorganism showed a tendency of increasing from spore formation stage to primordium differentiation stage and reached the highest, and then decreasing the microflora numbers when the M. conica developed into the fruit-body growth stage, but numbers of fungi were relatively changed lower than others.Generally, the bacterial and fungi population increased significantly from spore-formation stage to the primordia formation stage and increased up, then decreased at fruit-body maturing stage while the actinomycetes did not show the same order.
Group with ammonium ferric sulfate [NH 4 Fe(SO 4 ) 2 .12H 2 O] often had the highest number compared to others at different stages.So, these results suggested that numbers of microorganisms of the grown morel soils differ in different ferric fertilizers.From these results, we also think that it is necessary to use the denaturing gradient gel electrophoresis (DGGE) technique with environmental samples to analyze the genetic diversity of microflora populations, especially in DGGE study of fungal communities (Vainio and Hantula, 2000;Anderson et al., 2003).

Determination of enzymatic activity for Morchella grown soil
Soils were sampled from three stages which morel grows.At all sites there was a significant treatment effect for each enzyme tested (P<0.05).The data suggested that four soil enzyme activities increased with the morel growing and reaches a maximum at primordia formation stage.M. conica grown soil with the ferric fertilizers' disposal at different stages had different effects on four soil enzyme (urease, polyphenol oxidase, invertase and protease) (Table 2).From the results, we found that four soil enzymatic activities increased with the morel growing and reaches a maximum at primordium differentiation stage.Among the four soil enzymes, polyphenol oxidase, invertase and protease showed a higher activity than urease.Group with ammonium ferric sulfate showed higher urease activity than those of other groups, probably due to the organic material or the greater nitrogen inputs and sub-sequent stimulation of microbial activity.Experimental groups showed higher activities in soil enzymes when compared to the control on a large scale.The data suggests that four soil enzyme activities increased with the morel growing and reaches a maximum at primordia formation stage.Among the four soil enzyme, polyphenol oxidase, invertase and protease showed a higher activity than urease.
During spore-formation stage, soil urease activities showed a tendency with NH 4 Fe(SO 4 ) 2 .12H 2 O>FeSO 4 •7H 2 O>Fe 2 (SO 4 ) 3 •9H 2 O>Fe• EDDHA and Ferric citrate> Nutrients>ck, and significant difference were shown between experimental groups except Nutrients and control group (p<0.05),especially the group treated with ammonium ferric sulfate[NH 4 Fe(SO 4 ) 2 .12H 2 O] had been increased 8 times than control (p<0.01).Soil polyphenol oxidase activities of the experimental groups, except the group treated with Fe•EDDHA, showed significant differences when compared with that of the control.Invertase activities of the experimental groups were changed a little but not significantly different when compared with the control, while that of groups treated with FeSO 4 •7H 2 O and Nutrients were significantly decreased.Soil protease activities of the groups treated with Nutrients, NH 4 Fe(SO 4 ) 2 .12H 2 O and Fe•EDDHA were significantly increased when compared with that of the control.These results concludes that M. conica grown soil's enzymatic activities were effected by different ferric fertilizers, however, ammonium ferric sulfate (NH 4 Fe(SO 4 ) 2 .12H 2 O) and Fe•EDDHA relatively showed higher soil enzymatic activities than others.
Urease catalyzes the hydrolysis of urea into ammonia or ammonium ion depending on soil pH, and carbon dioxide.Urease is the most prominent involved in soil N cycling in the four enzymes (Tabatabai and Bremner, 1972;Cookson, 1999).Among the different enzymes in soil, dehydrogenase, urease and phosphatases are important in the transformation of plant nutrients (Gao et al., 2010).We know very well that morels, like other edible macrofungi, their coarse proteins, nitrogen uptake levels were influenced by soil urease and protease.Such results perhaps indicated increased soil urease activities was necessary in order to promote the growth of morel hyphae, accumulate the yield of morel fruiting bodies and lower yield of commercial cultivation of M. conica which is as a result of the lower level of nitrogen application.

Results of the commercial planting for M. conica
In this study, the epigeal fructifications (ascocarps) of M. conica are usually appeared in late February or in early March when soil temperature warms to above 10°C and this is in accordance with previous reports (Kanwal and Reddy, 2012;Masaphy, 2010;Goldway et al., 2000;Pilz et al., 2007;Ower et al., 1986).A number of studies point out that obtaining sclerotia are a necessary part of the process for domestication of the genus and commercial production (Buscot, 1994).Also, we found obvious stage of sclerotial formation in our cultivation period of M. conica.
In this study, each group fruiting season length (d) was also used to characterize morel fruit body production.Most groups seemed to harvest in 18-22 days, which showed fruiting season length was not significantly correlated with any fertilizer, still positively affected by the air temperature and soil temperature (Mihaila et al., 2007).The yield results are shown in Figures 3 and 4.
In our experiment, most experimental groups, except the group with ferric sulfate [Fe 2 (SO4) 3 .9H 2 O], increased the yields of M. conica; especially the groups with Fe•EDDHA and ammonium ferric sulfate [NH 4 Fe(SO 4 ) 2 .12H 2 O] resulted in higher yields of fruit-bodies than other experimental groups and higher than that of control group, 1.77 and 1.88 times of the control yields, respectively.We found that during primordial formation of morel, groups treated with ferric fertilizers appeared faster than the control, approximately three or four days earlier, with a highest soil microbial biomass and enzymatic activities.Maybe M. conica is responding to the different strategies Urease, [NH3-N/ (mg g -1 24 -1 )]; polyphenol oxidase, [gallic acid/(mg g -1 24 -1 )]; invertase, [glucose/(mg g -1 24 -1 )]; protease [amino acid/(mg g -1 24 -1 )].
for growth and sclerotia formation.The result of better morel growth might be with the increasing soil microbial population due to fertilizer application, higher release of root exudates and larger amounts of root residue left in the soil, which positively influence microbial processes and development.This indicates that different ferric fertilizers could influence the primordial formation of morel and affect the yield of fruiting bodies by its soil microbial population and enzymatic activities.
In nature, morel fruiting body formation is associated with a broad range of environmental stress conditions, some of which have yet to be defined (Masaphy, 2010) and up to now, there is no report about morel fruiting bodyformation and yield is associated with microelements, such as ferric, zinc, copper, molybdenum, among others.However, many literatures reported that microelements influenced the sclerotial formation and morels are rich in trace elements in their fruit-body (Kanwal and Reddy, 2012;Kalač, 2010;Amir et al., 1995).Fe•EDDHA is usually used to correct iron chlorosis in Strategy I plant, because of its great stability and solubility at high pHs (Rojas et al., 2008) 2008).In our study, it shows a higher effect on morel fruiting body than other ferric fertilizers did; the possible reasons may come from its great solubility.Nevertheless, the functional mechanisms of effects on different ferric fertilizers or other nutrition elements need to be further researched.In addtion, we think the same microelement or mineral element may influence the nutrient uptake for edible fungi by different approaches, it is difficult to give a sole conclusion whether a nutrient element promotes or hinders the experimental fungi to growth or even influence its yield at last.The best bet to optimize the nutrition plan for planting edible fungi especially Morchella spp. is to select a lot of nutrients (including trace elements) and to analyses their actual efficacy.

Conclusion
This paper reports studies on the effect of ferric fertilization on microbial biomass, enzymatic activities and morel yield.Sequencing of ITS showed that the species is closely related to M. conica.The results showed that among the six ferric fertilizers treatments, Fe•EDDHA, the ammonium ferric sulfate [NH 4 Fe(SO 4 ) 2 .12H 2 O] results in the highest amount of microbial biomass , enzymatic activities and morel yields, while the treatments of CK gave the opposite results.China is rich in nearly 30 morels species with eleven newly discover nine species within the Esculenta clade (yellow morels) and two novel species within the Elata clade (black morels), Over the 30 species, 20 appear to be endemic.The annual export of dried morels has been increased fivefold over the past 5 years to 900,000 kg, averaging $160 US dollars per kilogram (Du et al., 2012a).With their ever-increasing popularity, it is possible to purchase dried morels in local supermarkets in numerous countries throughout the year.Up to the present day, we have well known M. conica of its stable and effective planting condition.The study indicates ferric fertilizers making a big difference on the primordial formation of morel, further attempts regarding optimizing nutrition plan need to be carried out.
Figure 1.Mycelium and sclerotia observation by Scanning emission microscope for Morchella conica.Figure1A, mycelium of M. conica observed by SEM with the mycelium shaped as a kind of tube coated with small particles.Barrel-shaped cell in sclerotia.Bar= 1 μm.Figure1B, sclerotia of M. conica were about 15±3.24 μm in diameter, and its mycelium about 10.25±2.13μm in diameter.Bar= 1 μm.Figure1C, round, closely packed basal sclerotia.

Figure 3 .
Figure3.Artificial cultivation of morels.Figure3A, milky sclerotia produced by M. conica, after incubated to puffed wheat soil media; Figure3B, seed-planting.Morchella growing situation after hybridization, the plastic packages were equipped with the sterilized mixture including wheat grain, sawdust, chaff and vermiculite.They were used until the mycelium grown well; Figure3C, morel growing period, is relative to certain temperature and humidity.Fruiting season length is not significantly correlated with any fertilizer; Figure3D, fruit bodies in harvest time.The mature fruiting body reached 8-16 cm in length.The phenotypic features-fruiting body shape, color and size, are vary with, and affected by environmental conditions and developmental dynamics.

Table 1 .
Numbers of major soil microbial population groups from the Morchella grown soil.Mean values as M±SD.Within three mean values of each character, there is no significant differences between those contain same letters, and different letters in the same column denote significant differences among treatments according to ANOVA and LSD's multiple comparisons (p<0.05). a

Table 2 .
Soil enzymatic activities of the Morchella grown soil which treated by different ferric fertilizers.