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Research ArticleColonization and Diversity of AM Fungi by MorphologicalAnalysis on Medicinal Plants in Southeast China

Mingyuan Wang and Pan Jiang

Department of Horticulture Huaqiao University Xiamen 361021 China

Correspondence should be addressed to Mingyuan Wang w mingyuan163com

Received 10 September 2014 Revised 30 December 2014 Accepted 5 January 2015

Academic Editor Antonio Roldan Garrigos

Copyright copy 2015 M Wang and P Jiang This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The arbuscular mycorrhizal (AM) fungal distributions in the rhizosphere of 20 medicinal plants species in Zhangzhou southeastChina were studied The results showed 66 species of 8 genera of AM fungi were identified of which 38 belonged to Glomus 12to Acaulospora 9 to Scutellospora 2 to Gigaspora 2 to Funneliformis 1 to Septoglomus 1 to Rhizophagus and 1 to ArchaeosporaGlomus was the dominant genera and G melanosporum Acaulospora scrobiculata G etunicatum Funneliformis mosseae and Grubiformewere the prevalent speciesThe highest colonization (100) was recorded inDesmodium pulchellum (L) Benth while thelowest (80) was in Acorus tatarinowii Schott The AM fungi spore density ranged from 270 to 2860 per 100 g soil (average 1005)and the species richness ranged from 3 to 14 (average 97) per soil sample Shannon-Wiener index ranged from 052 to 2 (average145) In the present study the colonization had a highly negative correlation with available K and electrical conductivity Speciesrichness correlated positively with electrical conductivity and organic matter Shannon-Wiener index had a highly significantnegative correlation with pH This study provides a valuable germplasm and theoretical basis for AM fungal biotechnology onmedicinal standardization planting

1 Introduction

Arbuscular mycorrhizal (AM) fungi the most ubiquitoussymbiosis in nature are a kind of these soil microbesReports suggest that estimated 80 of plant species formsmycorrhizas [1] In general AM fungi and the host plantsare reciprocal symbionts The symbiosis improves plants thenutrient uptake and provides protection from pathogenswhile the AM fungi receive carbohydrates [2ndash4]

All over the world 80 of the rural population indeveloping countries utilizes locally medicinal plants forprimary healthcare And in China the use of different partsof medicinal plants to cure specific illness has been popularfrom ancient time In Zhangzhou southeast China thetypical humid subtropical monsoon climate contributes tothe growth of more than 700 kinds of lush medicinal plantsand creates unique ecological conditions for species diversityand distribution of AM fungi

The distribution of AM fungi associated with medicinalplants has been reported In a survey on AM association with

three different endangered species of Leptadenia reticulataMitragyna parvifolia andWithania coagulans high diversityof AMF was observed and Glomus constrictum Glomus fas-ciculatum Glomus geosporum Glomus intraradices Glomusmosseae and Glomus rubiforme were the most dominantspecies [5] Similarly 34 AM fungal species were identifiedfrom 36 medicinal plant species [6] Approximately 15 fungalspecies from 10 genera were isolated from the collected soilsin medicinal plant species lemon balm (Melissa officinalisL) sage (Salvia officinalis L) and lavender (Lavandulaangustifolia Mill) [7] About 50 species of medicinal plantsfrom 19 families have been studied in the associationwithAMfungi [8]

However not enough has been focused on the mycor-rhizal association withmedicinal plants Generally AM fungispecies in different ecosystems are affected by edaphic factorsso it is necessary to investigate the spatial distribution andcolonization of AM fungi related to the medicinal plants[9ndash13] Hence the present study is attempted to investigate

Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 753842 7 pageshttpdxdoiorg1011552015753842

2 The Scientific World Journal

Table 1 The botanic families response to the 20 medicinal plants

Latin name Botanic familyWoodwardia japonica (L f) Sm BlechnaceaeMelastoma candidum DDon RanunculaceaeLeonurus heterophyllus Sweet f LabiataeOcimum gratissimum L var suave (Willd) Hookf LabiataeDesmodium pulchellum (L) Benth PapilionaceaeLygodium japonicum (Thunb) Sw LygodiaceaeMentha haplocalyx Briq LabiataeGonostegia hirta (Blume) Miq UrticaceaeGardenia jasminoides Ellis RubiaceaeMallotus apelta (Lour) MuellArg EuphorbiaceaeAntenoron filiformeThunb PolygonaceaePolygonum chinense L PolygonaceaeSarcandra glabra (Thunb) Nakai ChloranthaceaePogonatherum crinitum (Thunb) Kunth AgrostidoidaceaeSelaginella uncinata (Desv) Spring SelaginellaceaeLophatherum gracile Brongn AgrostidoidaceaeAlpinia officinarumHance ZingiberaceaeAcorus tatarinowii Schott AraceaeArdisia crenata Sims MyrsinaceaeCitrus medica L var sarcodactylis Swingle Rutaceae

the diversity of AM fungi associated with medicinal plantspecies in Zhangzhou southeast China

2 Materials and Methods

21 Study Sites The city of Zhangzhou Fujian province asubtropical region is located on 23∘081015840ndash25∘061015840N and 116∘531015840ndash118∘091015840E The mean annual temperature is 21∘C with yearlyprecipitation of 1000ndash1700mmand annual sunshine of 2000ndash2300 hours Frost-free periods add up to more than 330 dayswith cool summer and warm winter The medicinal plantsin this study were collected from Xiaoxi town (24∘441015840N118∘171015840E) which was cinnamon soil from farmland andGuoqiang village (24∘351015840N 117∘561015840E) which was cinnamonsoil from woodland in Zhangzhou

22 Sample Collection The plants grew under natural envi-ronmental conditions Six healthy individuals per plantspecies of medicinal plants (Table 1) were randomly selectedfor the collection of rhizospheric soil and root samples180 soil and root samples were collected from Xiaoxi townand Guoqiang village in October 2011 For each plant threerandom soil cores at the depth of 0ndash30 cm about 1000 gwere established by contacting from the 6 duplicate plantsApproximately 20 plants species and 120 soil samples werecollected in total The subsamples were air-dried for 2 weeksand stored in sealed plastic bags at 4∘C for the followinganalysis

23 Estimation of AMColonization Themixed soil and rootssamples of each plant species were packed in polyethylenebags labeled and brought to the laboratory The soil samples

were air-dried at room temperature Roots were washed toremove soil particles preserved with FAA For colonizationmeasurement roots were cleared in 10 (wv) KOH andplaced in a water bath (90∘C) for 20ndash30minThe cooled rootsamples were then washed with water and stained with 05(wv) acid fuchsin Fifty root fragments for each sample (ca1 cm long)weremounted on slides in a polyvinyl alcohol solu-tion [14] and examined for the presence of AM structures at100ndash400x magnification with an Olympus BX50 microscopefor the presence of AM structures The percentage of rootcolonization was calculated using the following formula

root colonization ()

=Number of arbuscular mycorrhiza positive segments

Total number of segments studied

times 100(1)

24 AM Fungus Spore Quantification and IdentificationThree aliquots of soil (20 g) were obtained for every plantspecies AM fungal spores were extracted from the soil sam-ples by wet sieving and sucrose density gradient centrifuga-tion [15] Spores were counted under a dissectingmicroscopeand spore densities (SD) were expressed as the number ofspores per 100 g of soil The isolated spores were mountedin polyvinyl lactoglycerol (PVLG) Morphological identifica-tion of spores up to species level was based on spore sizecolor thickness of the wall layers and the subtendinghyphae by the identification manual [16] and the website ofthe International collection of vesicular and AM fungi(httpinvamwvuedu)

25 Soil Analysis Soil samples were air-dried and sievedthrough 2mm grid Three rhizospheric soil samples (le2mmfraction) for eachmedicinal plant were analyzed for their pHelectrical conductivity (EC) organic matter (OM) contentavailable N (N) available P (P) and available K (K) SoilpH was measured in soil water suspension 1 2 (wv) bypH meter (PHS-3C Shanghai Lida Instrument Factory) ECwas measured at room temperature in soil suspension (1 5wv) using conductivity meter (DDS-11C Shanghai HongYi instrument company) OM content was determined bythe Walkley-Black acid digestion method P (extracted with003M NH

4F-002M HCl) was measured by molybdenum

blue colorimetry K by an ammonium acetate method using aflame photometer and N by the alkaline hydrolysis diffusionmethod [17]

26 Diversity Studies Ecological measures of diversityincluding spore density (SD) species richness (SR) isolationfrequency (IF) Shannon-Wiener index (119867) and evenness (119869)were used to describe the structure of AM fungi communities[18 19] Diversity studies were carried out from Zhangzhouseparately for abundance and diversity of AM fungal speciesSpore density was defined as the number of AM fungi sporesand sporocarps in 100 g soil species richness wasmeasured asthe number of AM fungi species present in soil sample

The Scientific World Journal 3

Table 2 The rhizospheric soil properties of 20 medicinal plants

Host plants (20 species) P(mg kgminus1) K(mg kgminus1) N(mg kgminus1) OM(g 100 gminus1) pH EC(120583s cmminus1)Woodwardia japonica (L f) Sm 2358 plusmn 267 10478 plusmn 931 2238 plusmn 149 6550 plusmn 437 536 plusmn 036 5448 plusmn 363Melastoma candidum DDon 1842 plusmn 209 4388 plusmn 390 1493 plusmn 099 1097 plusmn 726 484 plusmn 032 8245 plusmn 550Leonurus heterophyllus Sweet f 70579 plusmn 7999 9560 plusmn 850 2396 plusmn 160 549 plusmn 366 778 plusmn 052 6255 plusmn 417Ocimum gratissimum L var suave(Willd) Hookf 11765 plusmn 1333 6491 plusmn 576 2213 plusmn 148 647 plusmn 431 552 plusmn 037 4345 plusmn 290

Desmodium pulchellum (L) Benth 1398 plusmn 158 5799 plusmn 515 11148 plusmn 743 1123 plusmn 749 662 plusmn 044 3540 plusmn 236Lygodium japonicum (Thunb) Sw 2268 plusmn 257 3948 plusmn 351 1556 plusmn 104 596 plusmn 397 550 plusmn 036 2815 plusmn 188Mentha haplocalyx Briq 97994 plusmn 11106 8386 plusmn 745 2933 plusmn 196 1549 plusmn 1033 629 plusmn 042 7295 plusmn 487Gonostegia hirta (Blume) Miq 1696 plusmn 192 4809 plusmn 427 2522 plusmn 168 1034 plusmn 689 474 plusmn 031 5875 plusmn 392Gardenia jasminoides Ellis 4385 plusmn 497 3932 plusmn 350 3641 plusmn 243 1008 plusmn 672 541 plusmn 036 4135 plusmn 276Mallotus apelta (Lour) MuellArg 3413 plusmn 386 3517 plusmn 313 2851 plusmn 190 809 plusmn 539 530 plusmn 035 4200 plusmn 280Antenoron filiformeThunb 20589 plusmn 2333 6541 plusmn 581 2993 plusmn 200 1017 plusmn 678 543 plusmn 036 5235 plusmn 349Polygonum chinense L 5030 plusmn 570 6490 plusmn 577 2505 plusmn 167 1008 plusmn 671 544 plusmn 029 4910 plusmn 327Sarcandra glabra (Thunb) Nakai 1848 plusmn 209 5629 plusmn 500 2238 plusmn 149 1111 plusmn 741 502 plusmn 033 6620 plusmn 441Pogonatherum crinitum (Thunb)Kunth 1046 plusmn 119 2864 plusmn 255 1551 plusmn 103 874 plusmn 583 527 plusmn 035 3570 plusmn 238

Selaginella uncinata (Desv) Spring 2329 plusmn 264 7183 plusmn 638 2391 plusmn 159 1045 plusmn 697 550 plusmn 037 3690 plusmn 246Lophatherum gracile Brongn 1804 plusmn 204 4746 plusmn 422 2753 plusmn 184 1084 plusmn 723 492 plusmn 032 4635 plusmn 309Alpinia officinarumHance 7451 plusmn 844 3676 plusmn 327 4202 plusmn 280 1329 plusmn 886 460 plusmn 030 8515 plusmn 567Acorus tatarinowii Schott 24054 plusmn 2726 18481 plusmn 1643 3931 plusmn 262 1094 plusmn 729 545 plusmn 039 25975 plusmn 1732Ardisia crenata Sims 2629 plusmn 298 4322 plusmn 384 4763 plusmn 318 1444 plusmn 963 466 plusmn 042 6690 plusmn 489Citrus medica L var sarcodactylisSwingle 18199 plusmn 2063 7094 plusmn 631 4771 plusmn 334 1287 plusmn 858 592 plusmn 051 9300 plusmn 820

P available P K available K N available N OM organic matter EC electrical conductivity means of six replicates plusmn standard deviation

isolation frequency (IF) = (number of samples in whichthe species or genus was observedtotal samples) times 100Species diversity was assessed by the Shannon-Weiner indexas follows119867 = minussum119896

119894=1

(119875119894ln119875119894) species evenness is calculated

by the following formula 119869 = 119867119867max where119867max = minus ln 119878119878 = total number of species in the community (richness) 119875

119894is

the relative abundance of each identified species per samplingsite and is calculated by the following formula 119875

119894= 119899119894119873

where 119899119894is the spore numbers of a species and119873 is the total

number of identified spore samples 119867max is the maximal 119867and calculated by the following formula119867 = ln 119878 where 119878 isthe total number of identified species per sampling site

27 Statistical Analysis The analysis of Pearson correlationcoefficient variance (ANOVA) and principal componentwere all carried out with SPSS Bass 180 (SPSS Inc USA)ThePearson correlation coefficient was employed to determinethe relationships between AM colonization SD SR IF119867 119869and soil parameters Differences in soil parameters coloniza-tion SD SR IF119867 and 119869 were tested using one-way ANOVAand means were compared by least significant difference at5 level

3 Results

31 Soil Parameters Results of the rhizospheric soil param-eters of the 20 medicinal plants harvested at both sites are

summarized in Table 2 The soil P ranged from 1046mgkgminus1 to 97994mg kgminus1 the soil K from 2864mg kgminus1 to18481mg kgminus1 and the soil N from 1493mg kgminus1 to 11148mgkgminus1 The OM ranged from 549 g kgminus1 to 1444 g kgminus1 Fur-thermore the soil was acidic as the pH ranged from 460 to778 EC was 2815 120583s cmminus1 to 25975 120583s cmminus1

32 AM Colonization Diversity Index and Diversity of AMFungi Colonization rate SD SR119867 and 119869 of AM fungi in therhizosphere of 20 medicinal plants species are presented inTable 3 The percentage of root colonization ranged from 8to 100 with an average of 5899 The highest colonizationwas observed in Desmodium pulchellum (L) Benth andlowest in Acorus tatarinowii Schott The SD in associationwith the 20 medicinal plant species ranged from 270 to 2860spores per 100 g soil with an average of 1005 spores per 100 gsoil The highest SD was observed in the rhizospheric soil ofLophatherum gracile Brongn and significantly different within Leonurus heterophyllus Sweet f The highest SR (14) wasrecorded in Acorus tatarinowii Schott while the lowest (3)appeared in Leonurus heterophyllus Sweet f with a mean of968Themaximum119867 occurred inAcorus tatarinowii Schott(200) and the minimum in Leonurus heterophyllus Sweet f(052) (average 145) The 119869 of AM fungi ranged from 027 to096 (average 066)

The results showed that 66 species of 8 genera of AMfungi were isolated and identified of which 38 belonged to


Table 3 The AM colonization and diversity index of 20 species of medicinal plants

Host plant Colonization () SD100 g soil SR 119867 119869

Woodwardia japonica (L f) Sm 19 plusmn 173 670 plusmn 7444 13 plusmn 141 155 plusmn 014 060 plusmn 005Melastoma candidum DDon 27 plusmn 246 1090 plusmn 12111 7 plusmn 078 136 plusmn 012 070 plusmn 006Leonurus heterophyllus Sweet f 12 plusmn 109 270 plusmn 30 3 plusmn 023 052 plusmn 005 048 plusmn 004Ocimum gratissimum L var suave (Willd) Hookf 65 plusmn 592 680 plusmn 7556 10 plusmn 111 179 plusmn 016 078 plusmn 007Desmodium pulchellum (L) Benth 100 plusmn 911 1330 plusmn 14778 11 plusmn 123 117 plusmn 010 049 plusmn 004Lygodium japonicum (Thunb) Sw 40 plusmn 364 760 plusmn 8487 11 plusmn 109 172 plusmn 015 072 plusmn 006Mentha haplocalyx Briq 85 plusmn 774 910 plusmn 10112 11 plusmn 099 157 plusmn 014 065 plusmn 006Gonostegia hirta (Blume) Miq 69 plusmn 629 700 plusmn 7778 10 plusmn 101 124 plusmn 011 054 plusmn 004Gardenia jasminoides Ellis 42 plusmn 383 1450 plusmn 16178 11 plusmn 122 161 plusmn 014 067 plusmn 005Mallotus apelta (Lour) MuellArg 64 plusmn 583 660 plusmn 7333 11 plusmn 131 184 plusmn 016 074 plusmn 006Antenoron filiformeThunb 86 plusmn 784 810 plusmn 90 9 plusmn 100 099 plusmn 009 047 plusmn 004Polygonum chinense L 97 plusmn 885 540 plusmn 60 6 plusmn 068 137 plusmn 012 079 plusmn 007Sarcandra glabra (Thunb) Nakai 64 plusmn 584 770 plusmn 8566 9 plusmn 089 166 plusmn 014 076 plusmn 008Pogonatherum crinitum (Thunb) Kunth 62 plusmn 565 360 plusmn 40 5 plusmn 055 154 plusmn 014 096 plusmn 008Selaginella uncinata (Desv) Spring 70 plusmn 638 990 plusmn 110 8 plusmn 085 127 plusmn 011 061 plusmn 005Lophatherum gracile Brongn 92 plusmn 838 2860 plusmn 31798 8 plusmn 078 121 plusmn 010 058 plusmn 005Alpinia officinarumHance 95 plusmn 866 1160 plusmn 12832 13 plusmn 154 195 plusmn 019 076 plusmn 007Acorus tatarinowii Schott 8 plusmn 073 680 plusmn 7567 14 plusmn 156 200 plusmn 021 076 plusmn 007Ardisia crenata Sims 60 plusmn 547 980 plusmn 10889 12 plusmn 112 198 plusmn 018 080 plusmn 007Citrus medica L var sarcodactylis Swingle 21 plusmn 191 2420 plusmn 26845 11 plusmn 108 064 plusmn 007 027 plusmn 002SD spore density SR spore richness119867 Shannon-Weiner index 119869 evenness means of six replicates plusmn standard deviation

Glomus 12 to Acaulospora 9 to Scutellospora 2 to Gigaspora2 to Funneliformis 1 to Septoglomus 1 to Rhizophagus and 1to Archaeospora (Table 4)

Based on IF Glomus melanosporum Acaulospora scro-biculata Glomus etunicatum Funneliformis mosseae andGlomus rubiformewere the prevalent AM fungi in decreasingorder (Table 4) Generally AM fungi with an IV greaterthan 50 were defined as dominant species So Glomusmelanosporum was the prevalent AM fungi with the highestIF (100)Glomuswas the dominant genuswith an IF (100)followed by Acaulospora IF (95)

33 Correlation Analysis As the soil characteristics may playa key role in the ecological distribution of AM fungi P K NOM pH and EC of the soil samples were investigated In thepresent study the colonization was negatively correlated withAK and EC but positively correlated with OM Spore densitywas positively correlated with OMThe same correlation wasfound between SR and N EC and OM 119867 was negativelycorrelated with pH whereas 119869 was negatively correlated withSD (Table 5)

4 Discussion

In the present study the composition and diversity of the AMfungi composition were described based on morphologicalspecies The results indicated that Glomus was the dominantgenus followed by Acaulospora Acaulospora and Glomusspecies usually produce more spores than Gigaspora andScutellospora species in the same environment [20 21]This may be explained by the difference in development

Acaulospora and Glomus species are thought to require lesstime to produce spores than Gigaspora and Scutellosporaspecies Furthermoremembers of theGigasporaceae typicallyestablish an extensive mycelium in soil and produce fewerspores than those of the Acaulosporaceae and Glomaceae[22 23]

The results showed a strong symbiotic relationshipbetween 20 medicinal plants and AM fungi but significantdifferenceswere observed in the different plant species As thestudies have shown nonrandom differences in distributionamong different AM fungi species and genera in the field itis also likely that the preferences of different AM fungi fordifferent host plants in our study might be reflected at thespecies or family level [24 25]

All 20 medicinal plants were infected by AM fungi butthe degree of colonization and the spore density varied amongplant species This may due to differences in the abilityof AM species to sporulate [26] The host plants used inthe trap cultures may also have been an important factorinfluencing mycorrhizal development spore formation anddistribution of AM fungi [27] Many AM species which infectthe roots of plants but do not sporulate in the soil mayhave remained undetected in the present study [28] Furtherstudies using molecular tools could solve this situation byallowing identification of AM fungi that colonize the rootsbut remain unsporulating

AM fungal SD SR have been positively correlated withOM OM could enhance spore production [29] extra radicalproliferation of hyphae [30] and improve AM colonization[31] In addition AM fungal hyphae grew best in soilswith a high amount of OM [32] Soil pH in our study


Table 4 Isolation frequency (IF) of AM fungi

Arbuscular mycorrhizal fungi species IFSeptoglomus 15 plusmn 110

Septoglomus constrictum Trappe 15 plusmn 161Archaeospora 10 plusmn 087

Archaeospora leptoticha Skenck amp Smith 10 plusmn 090Funneliformis 52 plusmn 478

Funneliformis mosseae (Nicolson amp Gerdemann)Gerdemann amp Trappe

50 plusmn 488

Funneliformis geosporum (Nicolson amp Gerdemann)Walker

45 plusmn 401

Gigaspora 15 plusmn 131Gigaspora margarita Becker amp Hall 5 plusmn 043Gigaspora ramisporophora Spain Sieverding ampSchenck

10 plusmn 097

Rhizophagus 15 plusmn 121Rhizophagus fasciculatum fasciculatum (Thaxt)Gerd amp Trappe

15 plusmn 140

Scutellospora 35 plusmn 323Scutellospora aurigloba (Hall) Walker amp Sanders 5 plusmn 044Scutellospora castaneaWalker 5 plusmn 039Scutellospora calospora Nicolson amp Gerd 5 plusmn 046Scutellospora erythropa Koske ampWalker 5 plusmn 061Scutellospora heterogama Nicolon amp Gerd 5 plusmn 048Scutellospora nigra (Redhead) Walker amp Sanders 5 plusmn 043Scutellospora pellucida Koske ampWalker 5 plusmn 045Scutellospora persica Koske ampWalker 5 plusmn 053Scutellospora rubra Sturmer amp Morton 5 plusmn 051

Acaulospora 95 plusmn 823Acaulospora bireticulata Rothwell amp Trappe 20 plusmn 178Acaulospora cavernata Blaszkowski 10 plusmn 088Acaulospora delicataWalker Pfeiffer amp Bloss 5 plusmn 040Acaulospora demannii Schenck amp Nicolson 15 plusmn 131Acaulospora excavataWalker amp Mason 10 plusmn 091Acaulospora foveata Trappe amp Janos 10 plusmn 09440Acaulospora gedanensis Blaszkowki 5 plusmn 048Acaulospora lacunosaMorton 10 plusmn 089Acaulospora laevis Gerdemann amp Trappe 5 plusmn 045Acaulospora mellea Sieverding amp Howeler 5 plusmn 047Acaulospora rehmii Sieverding amp Toro 5 plusmn 039Acaulospora scrobiculata Trappe 70 plusmn 667

Glomus 100 plusmn 1134Glomus aggregatum Schenck amp Smith 15 plusmn 163Glomus albidumWalker amp Rhode 40 plusmn 396Glomus arenarium Blaszkowski Tadych amp Madej 10 plusmn 087Glomus aureum Oehl Wiemken amp Sieverding 10 plusmn 094Glomus badium Oehl Redecker amp Sieverding 20 plusmn 212Glomus brohultii Sieverd amp Herrera 5 plusmn 048Glomus callosum Sieverding 5 plusmn 048Glomus citricola Tang amp zang 5 plusmn 044

Table 4 Continued

Arbuscular mycorrhizal fungi species IFGlomus claroideum Trappe amp Gerdemann 5 plusmn 041Glomus clarum Nicolson amp Gerdemann 10 plusmn 098Glomus convolutum Gerd amp Trappe 40 plusmn 386Glomus coremiodes Redecker amp Morton 5 plusmn 043Glomus coronatum Giovannetti Avio amp Salutini 5 plusmn 041Glomus deserticola Trappe Bloss amp Menge 10 plusmn 093Glomus diaphanumMorton ampWalker 15 plusmn 145Glomus dimorphicum Boyetchko amp Tewari 5 plusmn 043Glomus dolichosporum Zhang Wang amp Xing 20 plusmn 174Glomus etunicatum Becker amp Gerdemann 50 plusmn 484Glomus formosanumWu amp Chen 5 plusmn 038Glomus globiferum Koske ampWalker 10 plusmn 087Glomus heterosporum Smith amp Schenck 15 plusmn 144Glomus hyderabadensis Rani Kunwar Prasad ampManoharachary

5 plusmn 050

Glomus intraradices Schenck amp Smith 15 plusmn 132Glomus lamellosum Dalpe Koske amp Tews 15 plusmn 124Glomus luteum Kennedy Stutz amp Morton 5 plusmn 040Glomus macrocarpum (Tul amp Tul) Berch amp Fortin 5 plusmn 042Glomus manihotisHoweler Sieverding amp Schenck 10 plusmn 109Glomus melanosporum Gerdemann amp Trappe 100 plusmn 934Glomus microaggegatum Koske Gemma amp Olexia 15 plusmn 122Glomus microcarpum Tul amp Tul 5 plusmn 045Glomus monosporum Trappe amp Gerd 5 plusmn 045Glomus multicaule Gerdemann amp Bakshi 15 plusmn 133Glomus reticulatum Bhattacharjee amp Mukerji 15 plusmn 145Glomus rubiforme (Gerd amp Trappe) Almeida ampSchenck

50 plusmn 466

Glomus sinuosum Almeida amp Schenck 5 plusmn 045Glomus verruculosum Blaszkowski amp Tadych 10 plusmn 096Glomus versiforme (Karsten) Berch 10 plusmn 083Glomus viscosum Nicolson 5 plusmn 043

Means of six replicates plusmn standard deviation

was negatively correlated with AM fungal 119867 Soil pH couldaffect sporulation spore germination [33] hyphal growthand root colonization [34] and reproduction and communitystructure of AM fungi [35] The range of pH from 55 to65 has been found to favour Glomus to sporulate moreabundantly in acid soils [33]

In the present study SR was positively correlated withEC High EC could directly affect the solutes on osmoticpotential and delay or prevent all or any of the sporegermination phases by dissolved salts in the soil solutionAs solution concentrations increased maximum percentgermination and germination rate declined Effects of salinityon photosynthesis are known to differ between plant speciesand also between plants at different stages of development[35]


Table 5 Correlation analysis between AM fungi and different edaphic factors

P K N OM pH EC Colonization SD SR 119867 119869

P 1000K 0393lowast 1000N minus0064 0056 1000OM 0160 minus0023 0391lowast 1000pH 0631lowastlowast 0343lowast 0292 minus0170 1000EC 0199 0792lowastlowast 0070 0268 minus0027 1000Colonization minus0113 minus0479lowastlowast 0283 0365lowast minus0208 minus0442lowastlowast 1000SD minus0164 minus0164 0306 0424lowastlowast minus0120 minus0017 014 1000SR minus0134 0250 0361lowast 0410lowastlowast minus0227 0381lowast minus0042 0236 1000119867 minus0265 minus0001 minus007 0200 minus0488lowastlowast 0240 0115 minus0216 0585lowastlowast 1000119869 minus0214 minus0122 minus027 0080 minus0289 0060 0141 minus0365lowast 0114 0795lowastlowast 1000P available P K available K N available N OM organic matter EC electrical conductivity SD spore density SR spore richnessH Shannon-Weiner index119869 evenness lowast119875 lt 005 lowastlowast119875 lt 001

The AM colonization and diversity of medicinal plantsin southeast China were investigated in the present studyFrom the research we could conclude that the biodiversityof AM fungi was abundant thoughGlomuswas the dominantgenusThedegree of colonization and the spore density variedmarkedly among plant species Considering the potentialapplication of AM fungi on medicinal plants it seems thatmore attention should be paid to the predominant AM fungiduring the process of their cultivation especially mycorrhizalperformance (eg improving growth increasing secondarymetabolite production)

Conflict of Interests

Mingyuan Wang and Pan Jiang declared that there is nocompeting interest regarding the publication of this paper

Acknowledgments

The work was supported by the National Natural ScienceFoundation of China (no 31101512) and the FundamentalResearch Funds for the Central Universities (no JB-ZR1149)

References

[1] BWang andY-LQiu ldquoPhylogenetic distribution and evolutionof mycorrhizas in land plantsrdquo Mycorrhiza vol 16 no 5 pp299ndash363 2006

[2] S E Smith and D J Read Mycorrhizal Symbiosis AcademicPress San Diego Calif USA 2008

[3] M J Mohammad W L Pan and A C Kennedy ldquoSeasonalmycorrhizal colonization of winter wheat and its effect onwheatgrowth under dryland field conditionsrdquoMycorrhiza vol 8 no3 pp 139ndash144 1998

[4] B N Bearden and L Petersen ldquoInfluence of arbuscular myc-orrhizal fungi on soil structure and aggregate stability of avertisolrdquo Plant and Soil vol 218 no 1-2 pp 173ndash183 2000

[5] J Panwar and J C Tarafdar ldquoDistribution of three endangeredmedicinal plant species and their colonization with arbuscularmycorrhizal fungirdquo Journal of Arid Environments vol 65 no 3pp 337ndash350 2006

[6] S Zubek J Błaszkowski and P Mleczko ldquoArbuscular mycor-rhizal and dark septate endophyte associations of medicinalplantsrdquo Acta Societatis Botanicorum Poloniae vol 80 no 4 pp285ndash292 2011

[7] S Zubek A M Stefanowicz J Błaszkowski M Niklinska andK Seidler-Łozykowska ldquoArbuscular mycorrhizal fungi and soilmicrobial communities under contrasting fertilization of threemedicinal plantsrdquoApplied Soil Ecology vol 59 pp 106ndash115 2012

[8] Y Zeng L-P Guo B-D Chen et al ldquoArbuscular mycorrhizalsymbiosis and active ingredients of medicinal plants currentresearch status and prospectivesrdquoMycorrhiza vol 23 no 4 pp253ndash265 2013

[9] T P Mcgonigle and M H Miller ldquoDevelopment of fungi belowground in association with plants growing in disturbed andundisturbed soilsrdquo Soil Biology amp Biochemistry vol 28 no 3pp 263ndash269 1996

[10] A-H Eom D C Hartnett and G W T Wilson ldquoHost plantspecies effects on arbuscular mycorrhizal fungal communitiesin tallgrass prairierdquoOecologia vol 122 no 3 pp 435ndash444 2000

[11] P Vandenkoornhuyse R Husband T J Daniell et al ldquoArbus-cular mycorrhizal community composition associated with twoplant species in a grassland ecosystemrdquoMolecular Ecology vol11 no 8 pp 1555ndash1564 2002

[12] C E Lovelock KAndersen and J BMorton ldquoArbuscularmyc-orrhizal communities in tropical forests are affected by host treespecies and environmentrdquo Oecologia vol 135 no 2 pp 268ndash279 2003

[13] A Gollotte D van Tuinen and D Atkinson ldquoDiversity ofarbuscular mycorrhizal fungi colonising roots of the grassspecies Agrostis capillaris and Lolium perenne in a field exper-imentrdquoMycorrhiza vol 14 no 2 pp 111ndash117 2004

[14] J M Phillips andD S Hayman ldquoImproved procedure for clear-ing roots and staining parasitic and vesicular-arbuscularmycor-rhizal fungi for rapid assessment of colonizationrdquo Transactionsof the British Mycological Society vol 55 no 1 pp 158ndash1611970

[15] F Oehl E Sieverding K Ineichen P Mader T Boller and AWiemken ldquoImpact of land use intensity on the species diversityof arbuscular mycorrhizal fungi in agroecosystems of CentralEuroperdquoApplied and EnvironmentalMicrobiology vol 69 no 5pp 2816ndash2824 2003

[16] httpschuessleruserwebmwndeamphylo


[17] A L Page RHMiller andD R KeeneyMethods of Soil Analy-sis American Society of Anesthesiologists Soil Science Societyof America Madison Wis USA 1982

[18] E H Simpson ldquoMeasurement of diversityrdquoNature vol 163 no4148 p 688 1949

[19] M Franke-Snyder D D Douds Jr L Galvez et al ldquoDiversity ofcommunities of arbuscular mycorrhizal (AM) fungi presentin conventional versus low-input agricultural sites in easternPennsylvania USArdquo Applied Soil Ecology vol 16 no 1 pp 35ndash48 2001

[20] J D Bever J B Morton J Antonovics and P A Schultz ldquoHost-dependent sporulation and species diversity of arbuscular myc-orrhizal fungi in a mown grasslandrdquo Journal of Ecology vol 84no 1 pp 71ndash82 1996

[21] S N Suresh andN Nagarajan ldquoBiodiversity of arbuscular myc-orrhizal fungi in evergreen vegetation of westernghatsrdquo Journalof Pure and Applied Microbiology vol 4 no 1 pp 415ndash4192010

[22] M M Hart and R J Reader ldquoTaxonomic basis for variation inthe colonization strategy of arbuscular mycorrhizal fungirdquoNewPhytologist vol 153 no 2 pp 335ndash344 2002

[23] J S Piotrowski T Denich J N Klironomos J M Grahamand M C Rillig ldquoThe effects of arbuscular mycorrhizas on soilaggregation depend on the interaction between plant and fungalspeciesrdquo New Phytologist vol 164 no 2 pp 365ndash373 2004

[24] T Helgason J W Merryweather J Denison P Wilson J P WYoung and A H Fitter ldquoSelectivity and functional diversity inarbuscularmycorrhizas of co-occurring fungi and plants from atemperate deciduous woodlandrdquo Journal of Ecology vol 90 no2 pp 371ndash384 2002

[25] R Husband E A Herre and J P W Young ldquoTemporal vari-ation in the arbuscular mycorrhizal communities colonisingseedlings in a tropical forestrdquo FEMS Microbiology Ecology vol42 no 1 pp 131ndash136 2002

[26] K Turnau P Ryszka V Gianinazzi-Pearson andD vanTuinenldquoIdentification of arbuscular mycorrhizal fungi in soils androots of plants colonizing zinc wastes in southern PolandrdquoMycorrhiza vol 10 no 4 pp 169ndash174 2001

[27] R Liu and FWang ldquoSelection of appropriate host plants used intrap culture of arbuscular mycorrhizal fungirdquo Mycorrhiza vol13 no 3 pp 123ndash127 2003

[28] H Tian J P Gai J L Zhang P Christie and X L LildquoArbuscular mycorrhizal fungi associated with wild forageplants in typical steppe of eastern Inner Mongoliardquo EuropeanJournal of Soil Biology vol 45 no 4 pp 321ndash327 2009

[29] D D Douds Jr L Galvez M Franke-Snyder C Reider andL E Drinkwater ldquoEffect of compost addition and crop rotationpoint upon VAM fungirdquo Agriculture Ecosystems amp Environ-ment vol 65 no 3 pp 257ndash266 1997

[30] E J Joner and I Jakobsen ldquoGrowth and extracellular phos-phatase activity of arbuscular mycorrhizal hyphae as influencedby soil organicmatterrdquo Soil Biology and Biochemistry vol 27 no9 pp 1153ndash1159 1995

[31] X L He S Mouratov and Y Steinberger ldquoSpatial distributionand colonization of arbuscular mycorrhizal fungi under thecanopies of desert halophytesrdquo Arid Land Research and Man-agement vol 16 no 2 pp 149ndash160 2002

[32] J E Frey and J R Ellis ldquoRelationship of soil properties and soilamendments to response of Glomus intraradices and soybeansrdquoCanadian Journal of Botany vol 75 no 3 pp 483ndash491 1997

[33] GMWangD P Stribley P B Tinker andCWalker ldquoEffects ofpH on arbuscular mycorrhiza I Field observations on the long-term liming experiments at Rothamsted and Woburnrdquo NewPhytologist vol 124 no 3 pp 465ndash472 1993

[34] C A B Medeiros R B Clark and J R Ellis ldquoGrowth andnutrient uptake of sorghum cultivated with vesicular-arbus-cular mycorrhiza isolates at varying pHrdquoMycorrhiza vol 4 no5 pp 185ndash192 1994

[35] G-H An S Miyakawa A Kawahara M Osaki and TEzawa ldquoCommunity structure of arbuscular mycorrhizal fungiassociatedwith pioneer grass speciesMiscanthus sinensis in acidsulfate soils habitat segregation along pH gradientsrdquo SoilScience and Plant Nutrition vol 54 no 4 pp 517ndash528 2008

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ClimatologyJournal of


Table 1 The botanic families response to the 20 medicinal plants

Latin name Botanic familyWoodwardia japonica (L f) Sm BlechnaceaeMelastoma candidum DDon RanunculaceaeLeonurus heterophyllus Sweet f LabiataeOcimum gratissimum L var suave (Willd) Hookf LabiataeDesmodium pulchellum (L) Benth PapilionaceaeLygodium japonicum (Thunb) Sw LygodiaceaeMentha haplocalyx Briq LabiataeGonostegia hirta (Blume) Miq UrticaceaeGardenia jasminoides Ellis RubiaceaeMallotus apelta (Lour) MuellArg EuphorbiaceaeAntenoron filiformeThunb PolygonaceaePolygonum chinense L PolygonaceaeSarcandra glabra (Thunb) Nakai ChloranthaceaePogonatherum crinitum (Thunb) Kunth AgrostidoidaceaeSelaginella uncinata (Desv) Spring SelaginellaceaeLophatherum gracile Brongn AgrostidoidaceaeAlpinia officinarumHance ZingiberaceaeAcorus tatarinowii Schott AraceaeArdisia crenata Sims MyrsinaceaeCitrus medica L var sarcodactylis Swingle Rutaceae

the diversity of AM fungi associated with medicinal plantspecies in Zhangzhou southeast China

2 Materials and Methods

21 Study Sites The city of Zhangzhou Fujian province asubtropical region is located on 23∘081015840ndash25∘061015840N and 116∘531015840ndash118∘091015840E The mean annual temperature is 21∘C with yearlyprecipitation of 1000ndash1700mmand annual sunshine of 2000ndash2300 hours Frost-free periods add up to more than 330 dayswith cool summer and warm winter The medicinal plantsin this study were collected from Xiaoxi town (24∘441015840N118∘171015840E) which was cinnamon soil from farmland andGuoqiang village (24∘351015840N 117∘561015840E) which was cinnamonsoil from woodland in Zhangzhou

22 Sample Collection The plants grew under natural envi-ronmental conditions Six healthy individuals per plantspecies of medicinal plants (Table 1) were randomly selectedfor the collection of rhizospheric soil and root samples180 soil and root samples were collected from Xiaoxi townand Guoqiang village in October 2011 For each plant threerandom soil cores at the depth of 0ndash30 cm about 1000 gwere established by contacting from the 6 duplicate plantsApproximately 20 plants species and 120 soil samples werecollected in total The subsamples were air-dried for 2 weeksand stored in sealed plastic bags at 4∘C for the followinganalysis

23 Estimation of AMColonization Themixed soil and rootssamples of each plant species were packed in polyethylenebags labeled and brought to the laboratory The soil samples

were air-dried at room temperature Roots were washed toremove soil particles preserved with FAA For colonizationmeasurement roots were cleared in 10 (wv) KOH andplaced in a water bath (90∘C) for 20ndash30minThe cooled rootsamples were then washed with water and stained with 05(wv) acid fuchsin Fifty root fragments for each sample (ca1 cm long)weremounted on slides in a polyvinyl alcohol solu-tion [14] and examined for the presence of AM structures at100ndash400x magnification with an Olympus BX50 microscopefor the presence of AM structures The percentage of rootcolonization was calculated using the following formula

root colonization ()

=Number of arbuscular mycorrhiza positive segments

Total number of segments studied

times 100(1)

24 AM Fungus Spore Quantification and IdentificationThree aliquots of soil (20 g) were obtained for every plantspecies AM fungal spores were extracted from the soil sam-ples by wet sieving and sucrose density gradient centrifuga-tion [15] Spores were counted under a dissectingmicroscopeand spore densities (SD) were expressed as the number ofspores per 100 g of soil The isolated spores were mountedin polyvinyl lactoglycerol (PVLG) Morphological identifica-tion of spores up to species level was based on spore sizecolor thickness of the wall layers and the subtendinghyphae by the identification manual [16] and the website ofthe International collection of vesicular and AM fungi(httpinvamwvuedu)

25 Soil Analysis Soil samples were air-dried and sievedthrough 2mm grid Three rhizospheric soil samples (le2mmfraction) for eachmedicinal plant were analyzed for their pHelectrical conductivity (EC) organic matter (OM) contentavailable N (N) available P (P) and available K (K) SoilpH was measured in soil water suspension 1 2 (wv) bypH meter (PHS-3C Shanghai Lida Instrument Factory) ECwas measured at room temperature in soil suspension (1 5wv) using conductivity meter (DDS-11C Shanghai HongYi instrument company) OM content was determined bythe Walkley-Black acid digestion method P (extracted with003M NH

4F-002M HCl) was measured by molybdenum

blue colorimetry K by an ammonium acetate method using aflame photometer and N by the alkaline hydrolysis diffusionmethod [17]

26 Diversity Studies Ecological measures of diversityincluding spore density (SD) species richness (SR) isolationfrequency (IF) Shannon-Wiener index (119867) and evenness (119869)were used to describe the structure of AM fungi communities[18 19] Diversity studies were carried out from Zhangzhouseparately for abundance and diversity of AM fungal speciesSpore density was defined as the number of AM fungi sporesand sporocarps in 100 g soil species richness wasmeasured asthe number of AM fungi species present in soil sample








119894=1



119894is


119894= 119899119894119873




3 Results












4 Discussion












50 plusmn 488


45 plusmn 401


10 plusmn 097


15 plusmn 140




Table 4 Continued


5 plusmn 050


50 plusmn 466












Acknowledgments


References









































Journal of












Volume 2014

Advances in









Geophysics





















119894=1



119894is


119894= 119899119894119873




3 Results












4 Discussion












50 plusmn 488


45 plusmn 401


10 plusmn 097


15 plusmn 140




Table 4 Continued


5 plusmn 050


50 plusmn 466












Acknowledgments


References









































Journal of












Volume 2014

Advances in









Geophysics





















4 Discussion












50 plusmn 488


45 plusmn 401


10 plusmn 097


15 plusmn 140




Table 4 Continued


5 plusmn 050


50 plusmn 466












Acknowledgments


References









































Journal of












Volume 2014

Advances in









Geophysics




















50 plusmn 488


45 plusmn 401


10 plusmn 097


15 plusmn 140




Table 4 Continued


5 plusmn 050


50 plusmn 466












Acknowledgments


References









































Journal of












Volume 2014

Advances in









Geophysics





















Acknowledgments


References









































Journal of












Volume 2014

Advances in









Geophysics






































Journal of












Volume 2014

Advances in









Geophysics


















Journal of












Volume 2014

Advances in









Geophysics














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Research Article Colonization and Diversity of AM Fungi by ...downloads.hindawi.com/journals/tswj/2015/753842.pdf · Research Article Colonization and Diversity of AM Fungi by Morphological