colonization and spore richness of arbuscular mycorrhizal...

Research ArticleColonization and Spore Richness of Arbuscular MycorrhizalFungi in Araucaria Nursery Seedlings in Curitiba Brazil

Carlos Vilcatoma-Medina 1 Glaciela Kaschuk 2 and Flaacutevio Zanette1

1Programa de Pos-Graduacao em Agronomia-Producao Vegetal Departamento de FitotecniaFederal University of Parana Curitiba PR Brazil2Programa de Pos-Graduacao em Ciencia do Solo Departamento de Solos e Engenharia AgrıcolaFederal University of Parana Curitiba PR Brazil

Correspondence should be addressed to Carlos Vilcatoma-Medina carlosvilcatomaufprbr

Received 19 October 2017 Revised 19 January 2018 Accepted 5 February 2018 Published 1 April 2018

Academic Editor Mathias N Andersen

Copyright copy 2018 Carlos Vilcatoma-Medina et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Araucaria or Parana pine [Araucaria angustifolia (Bertol) Kuntze 1898] is an endangered timber tree species of Atlantic Forestthat naturally forms symbiosis with arbuscular mycorrhizal fungi (AMF) The objective of this experiment was to evaluate AMFcolonization and spore AMF richness in araucaria seedlings produced in nursery at the metropolitan region of Curitiba Brazilwith the interest of identifying a taxonomical AMF group For that soil and fine roots of 6-month- 1-year- 2-year- 3-year- and5-year-old araucaria seedlings were sampled and evaluated Evaluations indicated that araucaria seedlings were well colonized byAMF (with rates varying from almost 50 to over 85) and produced an abundant number of mycorrhizal spores (from 344 to676 spores per seedling) Samples contained spores of the species Acaulospora scrobiculata Dentiscutata heterogama and Glomusspinuliferum and unidentified species of genera Gigaspora and Glomus The Glomus genus was the most abundant kind of AMFspores found under nursery conditionsTherefore the experiment evidenced that Glomus is a promising genus candidate for beingused as AMF inoculant in production of araucaria seedlings

1 Introduction

Araucaria or Parana pine [Araucaria angustifolia (Bertol)Kuntze 1898] is an endangered tree species of the MixedOmbrophilous Forest commonly known as ldquoMata dasAraucariasrdquo located in southern and the southern east ofBrazil and in portions of Argentina and Paraguay [1 2]The araucaria is a dioecious plant and its propagation byseeds (pinhoes) is conditioned to the existence of intraspecificgenetic richness in natural habitat The problem is that 97of the Mata das Araucarias ecosystem has disappeared [3]and reminiscent araucaria trees are isolated from each otherand may not form viable seeds In addition seeds are richin carbohydrates and proteins may be consumed by wildanimals and rapidly lose vigorwhen left on dry soil [4] Refor-estation by transplanting seed-formed seedlings is suitablestrategy but it takes too long before nursery seedlings reachadequate height for transplantation Yet adding fertilizer to

the substrate to increase growth ratesmay result in lower rootshoot ratio and consequently decrease the rates of survival inthe field after transplantation

Araucaria naturally establishes symbiosis with arbuscularmycorrhizal fungi (AMF) [4] in which the plant suppliesphotosynthates in exchange for competitive advantages ofgrowth and survival [5ndash8] Pioneer studies already reportedAMF in fine araucaria roots [9] however it was only recentlythat the AMF has been considered as a strategy to increasearaucaria growth under nursery conditions [10 11] In factthe studies performed so far show two important facts firstthat araucaria is colonized by a large and diverse number ofAMF species [10 12] and second that the araucaria positivelyrespond to inoculation of some AMF species both in highand low P supply [13] However we do not know precisely themost efficient and competitive AMF species under nurseryseedling production [14]

HindawiInternational Journal of AgronomyVolume 2018 Article ID 5294295 6 pageshttpsdoiorg10115520185294295

2 International Journal of Agronomy

Table 1 Chemical attributes of soils used in the nursery experiment with araucaria in Curitiba Brazil

Soil attributelowast Seedling age6 months 1 year 2 years 3 years 5-years

pH CaCl2 55 plusmn 0240 49 plusmn 0081 55 plusmn 0122 52 plusmn 0173 43 plusmn 0394Al3+ (cmoldm3) 00 plusmn 00 09 plusmn 0176 00 plusmn 00 00 plusmn 0042 27 plusmn 1494H + Al3+ (cmoldm3) 51 plusmn 0678 132 plusmn 1327 74 plusmn 0903 72 plusmn 0537 139 plusmn 2591Ca2+ (cmoldm3) 43 plusmn 0221 61 plusmn 0522 94 plusmn 0685 71 plusmn 1784 30 plusmn 1338Mg2+ (cmoldm3) 18 plusmn 0078 37 plusmn 0355 43 plusmn 0269 29 plusmn 0930 16 plusmn 1338K+ (cmoldm3) 11 plusmn 0260 03 plusmn 0151 10 plusmn 0286 07 plusmn 0443 02 plusmn 0128P (mgdm3) 167 plusmn 5390 782 plusmn 16967 1445 plusmn 16408 875 plusmn 30295 791 plusmn 42421C (gdm3) 127 plusmn 3457 853 plusmn 27451 643 plusmn 11419 467 plusmn 16486 614 plusmn 55447lowastpH measured in CaCl2 001mol Lminus1 Ca2+ Mg2+ and Al3+ extracted with KCl 1mol Lminus1 H+ + Al3+ extracted with calcium acetate 05mol Lminus1 K+ and Pextracted with the Mehlich-1 extractor C determined with K2Cr2O7

Therefore the objective of this experimentwas to evaluateAMF colonization and spore AMF richness in nursery soilsamples at the metropolitan region of Curitiba Brazil withthe interest of identifying the most abundant taxonomicalAMF groups that grow under those conditions

2 Material and MethodsAraucaria seedlings were produced from seeds in the nurseryof the Universidade Federal do Parana Curitiba (25∘2510158404710158401015840Sand 49∘1610158401910158401015840W 950m) Brazil under entirely randomdesign five replicates and five treatmentsThe substrate usedwas a mixture of soil and organic residues (crop residuesorganic compounds and others) collected in the metropoli-tan area of the city The experimental design was completelyrandomized with five replications in each treatment Theseedlings were at the moment of harvesting according tothe respective treatments 6-month 1-year 2-year and 5-year old Seedlings grew outdoors under full light naturalconditions and were well watered during the whole period ofexperiment The climate in the region is classified as KoppenCfb [15] and the eventual climate events such as frost andstorms did not damage any stage of plant growth

Soil samples were submitted to chemical analyses andthe pH was measured in CaCl2 001mol Lminus1 Ca2+ Mg2+and Al3+ were extracted with KCl 1M H+ + Al3+ withCa(C2H3O2)2 05M K+ and P with the Mehlich-1 extractorcarbon (C) with the K2Cr2O7 (Table 1)

Mycorrhizal spores were extracted with minor modifica-tions with the procedures of [16 17] as follows 100 g of soilsample was suspended in 100ml and left resting for 60minsoil suspensionswere liquefied for 10 s and sequentially sievedthrough 500 and 53 120583m mesh sieves spores were recoveredin tubes with 70 sucrose solution and centrifuged for fourmin twice and then sieved for 250 and 50120583m mesh sievesSpores with similar morphology were assembled then theywere spread on microscope slide and treated with polyvinylalcohol-lactic acid-glycerol [18] and the Melzer reagent [19]Spores were individually identified undermicroscope (PrimoStarZeiss) according to the morphological characteristicsusing the criteria of taxonomical classification proposed byInvam [20]

Root fragments were preserved in lactoglycerol (lacticacid glycerol and water in the proportion 1 2 1) before

100

80

60

40

20

0

Root

colo

niza

tion

()

c

ab abba

6 months 1 year 2 years 3 years 5 yearsAraucaria seedling age (year)

Figure 1 Root arbuscular mycorrhizal fungi (AMF) colonizationaraucaria seedlings with ages between 6 months and 5 years grownin a nursery in field in Curitiba Brazil Means followed by the sameletter are not significantly different according to Tukeyrsquos test (119901 le005)The error bars represent the standard error of themean 119899 = 5

measurements The rates of mycorrhizal colonization wereestimated by observing cleared and dyed root fragmentsunder microscope (Stemi 305Zeiss) considering a grid of1 cm times 1 cm [21] with minor modifications as follows fineroots were soaked in KOH 10 at room temperature during24 hours and then in water bath at 80∘C for an hour rootswere sequentially washed with H2O2 100 and water twiceand dyed with blue-pen ink in water bath at 80∘C for fiveminutes

Variance analyses and mean comparisons considered thestatistical design of entirely random design five replicatesand five treatments Redundancy analyses were performedwith Canoco for Windows 45 package 43 considering theabundance of spore taxonomic group and chemical attributesas the explanatory variables for the differences between thetreatments

3 Results

Araucaria seedlings with ages between 6 months and 5 yearsproduced under nursery conditions in Curitiba were wellcolonized by AMF (rates varying from almost 50 to over 85Figure 1) and contained an abundant number of mycorrhizal

International Journal of Agronomy 3

Table 2 Mean number of mycorrhizal spores of araucaria seedlings with ages between 6 months and 5 years grown in a nursery field inCuritiba Brazil

Mycorrhizal fungal species Seedling age (year)6 months 1 year 2 years 3 years 5 years

Acaulospora scrobiculata 13b 46ab 44ab 70a 33b

Dentiscutata heterogama 11a 16a 15a 17a 15a

Gigaspora sp 9b 29a 8b 12b 11b

Glomus spinuliferum 31b 114a 53b 129a 118a

Glomus sp 1 40ab 28b 40ab 62a 34ab

Glomus sp 2 91a 142a 55a 161a 141a

Glomus sp 3 101c 187a 82c 111bc 176ab

Glomus sp 4 48b 153a 57b 73b 148a

Total of species 344b 715a 355b 635a 676a

Means followed by the same letter in the lines do not differ by the Tukey test at 119901 lt 005

spores (varying from 344 to 676 Table 2) Samples containedspores of the species Acaulospora scrobiculata Dentiscutataheterogama and Glomus spinuliferum and of four unidenti-fied species of the generaGigaspora (one species) andGlomus(four different species)

Mycorrhizal fungi colonization increases from 40 toaround 80 in seedlings of 6 monthsndash1 year old From 1 yearonwards regardless of chemical differences in the soil usedrates of AMF colonization remained at levels of 80 over theyears At first sight there was a trend in which older seedlings(3- and 5-year old) containedmore spores than younger (eg6 months and 2 years old) seedlings but because 2-year oldaraucaria seedlings contained the largest number of sporesage was not the main factor affecting the number of spores inthis experiment

ThedominantAMF species in the 6-month 1-year 2-yearand 5-year old seedlings was Glomus sp 3 and in the 3 yearsold seedlings it wasGlomus sp 2 (Table 2)The least abundantspores belonged to the family Gigasporaceae (Dentiscutataheterogama and Gigaspora sp) and their numbers did notdiffer between treatments

Redundancy analyses showed that AMF spore richnessin 6-month old seedlings was not affected by soil chemi-cal attributes but it was so in older seedlings (Figure 2)Attributes related to potential soil acidity (Al+ and H+Al+)grouped AMF species Glomus sp 3 Glomus sp 4 andGigaspora sp Attributes related to good soil fertility (CaMg and P) grouped mycorrhizal fungi species Glomus sp1 Dentiscutata heterogama Acaulospora scrobiculata andGlomus sp 1 (Figure 2)

4 Discussion

Perennial plants with slow growth like araucaria are notcommonly taken into long-term experiments involving AMFbecause of the obvious difficulty of waiting for a long timefor the first results (eg compared to annual crops) andalso because araucaria is not the first choice of reforestationby companies Therefore although AMF colonization hasbeen studied long ago [9] our experiment demonstratedthat AMF colonization occurs under nursery conditions to

seedlings from very young age up to 5-year old seedlingswithout systematic inoculation and regardless of chemicalsoil attributes (Figure 1 Table 1)This implies that in additionto field natural conditions [10 22] araucaria seedlings are alsobenefited from AMF colonization under nursery conditions

A great limitation in the application of AMF symbiosisis the fact that we do not have established recommendationsof which AMF species are better adapted to nursery andfield conditions Previous studies indicated that araucaria iscapable of supporting AMF symbiosis with a greater numberof different fungal species [11 22ndash25] than that we foundin this experiment To date araucaria AMF spore richnessvaried from 8 [23] to 13 [11] in forests of the State of RioGrande do Sul and from 26 [24] to 58 [25] in forests ofSao Paulo The variation in spore AMF richness has beenfrequently attributed to heterogeneity in soil habitat and tothe variable capacity of AMF species to adapt to climate andsoil in different regions [10 13]Therefore although the AMFrichness is great under field conditions only few species areprobably well adapted to nursery conditions

The genusGlomus represented 86of the spores analyzedin our experiment (Table 2) Previously it was observed thatthis genus represented between 40 and 60 of AMF sporesin forests in the State of Sao Paulo [10 22] In fact soil acidityassociated with exchangeable soil Al (Al+) may affect sporegermination growth of germinative tube hyphae and root[26] Furthermore concentrations higher than 100 120583m Al+affect sporulation of certain species [27] In this experimentpotential acidity determined by high concentrations of Al+and H + Al+ was associated with Glomus sp 3 and Glomussp 4 (Figure 2) The Glomus genus is the most commonlyfound and probably the most adapted AMF to Brazilianacidy soils [28 29] This dominancy indicates that Glomusis highly competitive among native spores and it has a highreproductive capacity [10]

The Acaulospora genus was the second most abundantAMF spore in the experiment (Table 2) corroborating withthe fact that the Acaulospora AMF has been found inmoderate acidy soils (pH lower than 62) [30] and very acidyforest soils [24] The least abundant genus in this experimentbelonged to the genusGigaspora (Table 2)Gigasporahas been


RedundanciesAxis 1 Axis 2

063 minus018

minus043 031

minus072 014

018 minus052

minus008 minus040

064 minus014

minus007 minus055

minus044 minus025

6-month old1-year old2-year old

3-year old5-year old

10

10

Axi

s 2 2

06

minus10

minus10

Axis 1 569

Glo3

Glo4Ggsp

Glo2

Glof Dhtg

Ascr

Glo1

Al

C

Mg

P (ppm)

P (ppm)

Ca

K

（ + Ｆ

pH Ｆ2

Al (cmol＞Ｇ3)

（ + Ｆ (cmol＞Ｇ3)

Ca (cmol＞Ｇ3)

Mg (cmol＞Ｇ3)

K (cmol＞Ｇ3)

pH Ｆ2

C (g＞Ｇ3)

Figure 2 Redundancy analyses with several species of arbuscular mycorrhizal fungi in relation to chemical soil attributes in soil usedto produce araucaria seedlings in a nursery of Curitiba Brazil Note Ascr Acaulospora scrobiculata Dhtg Dentiscutata heterogama GgspGigaspora sp Glof Glomus spinuliferum Glo1 Glomus sp 1 Glo2 Glomus sp 2 Glo3 Glomus sp 3 Glo4 Glomus sp 4

associated with moderate acidy soils and high levels of phos-phate availability [13 14] In our experiment however Gigas-porawas not related to phosphate but it was slightly associatedwith potential soil acidity (Al+ and H + Al+ Figure 2)

The traditional paradigm describes regulation of AMFsymbioses in relation to phosphate availability in whichplants suppress AMF colonization once phosphate is avail-able in enough quantities for being taken from the soilsolution However this paradigm neglects some aspects ofAMF physiology Reference [31] gave evidence that afterthe AMF has colonized root wheat (Triticum aestivum L)plants it does not let it down even if phosphate is availablein enough quantities It does not imply necessarily thatAMF is always efficient regardless of P availability In factP availability determines different AMF compositions andtherefore different AMF efficiencies [32] Earlier studies thanthat of [31] demonstrated that plant growth responses toAMF inoculation under low and high phosphate availabilitydepend verymuch on the dominant AMF [33] In that aspectthe genus Glomus had a much greater phosphate uptake effi-ciency than Gigaspora [33] Interestingly greater efficiency ofGlomus over Gigaspora (increased mycorrhizal dependenceof plants) was alsomeasured on araucaria seedlings producedwith 150mg dmminus3 soil of phosphate fertilizer [14] Previousstudies indicated that mycorrhizal colonization occurs whenplants are grown in soils that contain less than 40mg dmminus3[32] In the present study the substrate contained 62mg ofP dmminus3 and even so Araucaria seedlings were well colonizedby mycorrhizae especially with the genus Glomus This is anobservation that should derive more research in our group

Considering the greater abundance of Glomus in relationto other genera our experiment suggested that other studiesshould search within Glomus diversity to identify AMF inoc-ulants that promote growth of araucaria seedlings The nextstep is selecting strains checking for consistent plant growthresponses and evaluating the viability in inoculant produc-tion at commercial scale As Glomus was not related to P soilconcentrations in soil samples (Figure 2) it is important thatselection occurs under reasonable soil chemical conditionsAs a matter of fact it has been shown that AMF symbiosesmay be favored by better plant growth [34] and high potentialacidity (high Al+) inhibits root growth damages cellularstructure and hampers water and nutrient [35 36]

5 Conclusions

Araucaria seedlings are intensively colonized by AMF atleast until the age of 5 years old even when they are notsystematically inoculated with specific AMF strains

Glomus genus is the most abundant kind of AMF sporesfound under nursery conditions and therefore other studiesshould search within Glomus diversity to identify AMFinoculants that promote growth of araucaria seedlings

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Authors thank Dr Celso Auer Dr Sidney Sturmer andDr Raul M Cesar respectively for the suggestions on


the experimental setup training on spore identificationand statistical analyses Carlos Vilcatoma-Medina receiveda CAPES (Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior) scholarship to pursue his PhD studies

References

[1] P Thomas ldquoAraucaria angustifoliardquo The IUCN Red List ofThreatened Species Article ID eT32975A2829141 2013

[2] G F Paludo M B Lauterjung M S dos Reis and A Man-tovani ldquoInferring population trends of Araucaria angustifolia(Araucariaceae) using a transition matrix model in an old-growth forestrdquo Southern Forests vol 78 no 2 pp 137ndash143 2016

[3] VM Stefenon O Gailing and R Finkeldey ldquoGenetic structureof Araucaria angustifolia (Araucariaceae) populations in Brazilimplications for the in situ conservation of genetic resourcesrdquoThe Journal of Plant Biology vol 9 no 4 pp 516ndash525 2007

[4] R B Zandavalli L R Dillenburg and P V D De SouzaldquoGrowth responses of Araucaria angustifolia (Araucariaceae)to inoculation with the mycorrhizal fungus Glomus clarumrdquoApplied Soil Ecology vol 25 no 3 pp 245ndash255 2004

[5] S Gianinazzi A Gollotte M-N Binet D van Tuinen DRedecker and DWipf ldquoAgroecology the key role of arbuscularmycorrhizas in ecosystem servicesrdquo Mycorrhiza vol 20 no 8pp 519ndash530 2010

[6] S E Smith and F A Smith ldquoRoles of arbuscular mycorrhizasin plant nutrition and growth New paradigms from cellular toecosystem scalesrdquo Annual Review of Plant Biology vol 62 pp227ndash250 2011

[7] S E Smith and F A Smith ldquoFresh perspectives on the rolesof arbuscular mycorrhizal fungi in plant nutrition and growthrdquoMycologia vol 104 no 1 pp 1ndash13 2012

[8] S C Jung A Martinez-Medina J A Lopez-Raez and MJ Pozo ldquoMycorrhiza-induced resistance and priming of plantdefensesrdquo Journal of Chemical Ecology vol 38 no 6 pp 651ndash664 2012

[9] F R Milanez and H Monteiro Neto ldquoNota previa sobremicorriza no pinho do Paranardquo Arquivos do Servico Florestalvol 4 pp 87ndash93 1950

[10] MMoreira-Souza S F B Trufem S M Gomes-Da-Costa andE J B N Cardoso ldquoArbuscular mycorrhizal fungi associatedwith Araucaria angustifolia (Bert) O KtzerdquoMycorrhiza vol 13no 4 pp 211ndash215 2003

[11] R B Zandavalli S L Sturmer and L R Dillenburg ldquoSpeciesrichness of arbuscular mycorrhizal fungi in forests with Arau-caria in Southern Brazilrdquo Hoehnea vol 35 no 1 pp 63ndash682008

[12] M Moreira M I Zucchi J E Gomes S M Tsai A Alves-Pereira and E J B N Cardoso ldquoAraucaria angustifolia above-ground roots presented high arbuscular mycorrhizal fungalcolonization and diversity in the Brazilian Atlantic ForestrdquoPedosphere vol 26 no 4 pp 561ndash566 2016

[13] M Moreira D Baretta and E J Cardoso ldquoDoses de fosforodeterminam a prevalencia de fungos micorrızicos arbuscularesem Araucaria angustifoliardquo Ciencia Florestal vol 22 no 4 pp813ndash820 2012

[14] MMoreira-Souza and E J Cardoso ldquoDependencia micorrızicade Araucaria angustifolia (Bert) O Ktze sob doses de fosforordquoRevista Brasileira de Ciencia do Solo vol 26 no 4 pp 905ndash9122002

[15] C A Alvares J L Stape P C Sentelhas J L De MoraesGoncalves and G Sparovek ldquoKoppenrsquos climate classificationmap for Brazilrdquo Meteorologische Zeitschrift vol 22 no 6 pp711ndash728 2013

[16] J Gerdemann and T H Nicolson ldquoSpores of mycorrhizalendogone species extracted from soil by wet sieving anddecantingrdquo Transactions of the British Mycological Society vol46 pp 235ndash244 1963

[17] W R Jenkins ldquoA rapid centrifugation technique for separatingnematodes from soilrdquo Plant Disease Report vol 48 article 6921964

[18] J B Morton S P Bentivenga andWWWheeler ldquoGerm plasmin the International Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (INVAM) and procedures forculture development documentation and storagerdquoMycotaxonvol 48 pp 491ndash528 1993

[19] R E Koske and B Tessier ldquoA convenient permanent slidemounting mediumrdquo Mycological Society of America Newslettevol 34 article 59 1983

[20] INVAM ldquoInternational culture collection of (vesicular) arbus-cular mycorrhizal fungirdquo httpsinvamwvuedu

[21] M Giovannetti and B Mosse ldquoAn evaluation of techniques formeasuring vesicular arbuscular mycorrhizal infection in rootsrdquoNew Phytologist vol 84 no 3 pp 489ndash500 1980

[22] M Moreira M A Nogueira S M Tsai S M Gomes-Da-Costa and E J B N Cardoso ldquoSporulation and diversity ofarbuscular mycorrhizal fungi in Brazil Pine in the field and inthe greenhouserdquoMycorrhiza vol 17 no 6 pp 519ndash526 2007

[23] M Breuninger W Einig E Magel E Cardoso and R HamppldquoMycorrhiza of Brazil pine (Araucaria angustifolia [Bert OKtze])rdquoThe Journal of Plant Biology vol 2 no 1 pp 4ndash10 2000

[24] M Moreira D Baretta S M Tsai and E J B N CardosoldquoArbuscular mycorrhizal fungal communities in native and inreplanted Araucaria forestrdquo Scientia Agricola vol 66 no 5 pp677ndash684 2009

[25] J A Bonfim R L F Vasconcellos T Gumiere D de LourdesColombo Mescolotti F Oehl and E J B Nogueira CardosoldquoDiversity of arbuscular mycorrhizal fungi in a BrazilianAtlantic Forest ToposequencerdquoMicrobial Ecology vol 71 no 1pp 164ndash177 2016

[26] A Seguel J R Cumming K Klugh-Stewart P Cornejo andF Borie ldquoThe role of arbuscular mycorrhizas in decreasingaluminium phytotoxicity in acidic soils a reviewrdquo Mycorrhizavol 23 no 3 pp 167ndash183 2013

[27] K Klugh-Stewart and J R Cumming ldquoOrganic acid exudationby mycorrhizalAndropogon virginicus L (broomsedge) roots inresponse to aluminumrdquo Soil Biology amp Biochemistry vol 41 no2 pp 367ndash373 2009

[28] N C Johnson and D A Wedin ldquoSoil carbon nutrientsand mycorrhizae during conversion of dry tropical forest tograsslandrdquo Ecological Applications vol 7 no 1 pp 171ndash182 1997

[29] C M R Pereira D K A D Silva A C D A Ferreira B TGoto andLCMaia ldquoDiversity of arbuscularmycorrhizal fungiin Atlantic forest areas under different land usesrdquo AgricultureEcosystems amp Environment vol 185 pp 245ndash252 2014

[30] S L Sturmer and M M Bellei ldquoComposition and seasonalvariation of spore populations of arbuscular mycorrhizal fungiin dune soils on the island of Santa Catarina BrazilrdquoBotany vol72 no 3 pp 359ndash363 1994

[31] H Li S E Smith R E Holloway Y Zhu and F A SmithldquoArbuscularmycorrhizal fungi contribute to phosphorus uptake


by wheat grown in a phosphorus-fixing soil even in the absenceof positive growth responsesrdquo New Phytologist vol 172 no 3pp 536ndash543 2006

[32] C D Collins and B L Foster ldquoCommunity-level consequencesof mycorrhizae depend on phosphorus availabilityrdquo Ecologyvol 90 no 9 pp 2567ndash2576 2009

[33] S E Smith F A Smith and I Jakobsen ldquoMycorrhizal fungican dominate phosphate supply to plants irrespective of growthresponsesrdquo Plant Physiology vol 133 no 1 pp 16ndash20 2003

[34] J A Mensah A M Koch P M Antunes E T Kiers MHart andH Bucking ldquoHigh functional diversity within speciesof arbuscular mycorrhizal fungi is associated with differencesin phosphate and nitrogen uptake and fungal phosphatemetabolismrdquoMycorrhiza vol 25 no 7 pp 533ndash546 2015

[35] L V Kochian M A Pineros J Liu and J V Magalhaes ldquoPlantadaptation to acid soils the molecular basis for crop aluminumresistancerdquoAnnual Review of Plant Biology vol 66 pp 571ndash5982015

[36] Z Rengel J Bose Q Chen and B N Tripathi ldquoMagnesiumalleviates plant toxicity of aluminium and heavy metalsrdquo Cropamp Pasture Science vol 66 no 12 pp 1298ndash1307 2015

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Table 1 Chemical attributes of soils used in the nursery experiment with araucaria in Curitiba Brazil

Soil attributelowast Seedling age6 months 1 year 2 years 3 years 5-years

pH CaCl2 55 plusmn 0240 49 plusmn 0081 55 plusmn 0122 52 plusmn 0173 43 plusmn 0394Al3+ (cmoldm3) 00 plusmn 00 09 plusmn 0176 00 plusmn 00 00 plusmn 0042 27 plusmn 1494H + Al3+ (cmoldm3) 51 plusmn 0678 132 plusmn 1327 74 plusmn 0903 72 plusmn 0537 139 plusmn 2591Ca2+ (cmoldm3) 43 plusmn 0221 61 plusmn 0522 94 plusmn 0685 71 plusmn 1784 30 plusmn 1338Mg2+ (cmoldm3) 18 plusmn 0078 37 plusmn 0355 43 plusmn 0269 29 plusmn 0930 16 plusmn 1338K+ (cmoldm3) 11 plusmn 0260 03 plusmn 0151 10 plusmn 0286 07 plusmn 0443 02 plusmn 0128P (mgdm3) 167 plusmn 5390 782 plusmn 16967 1445 plusmn 16408 875 plusmn 30295 791 plusmn 42421C (gdm3) 127 plusmn 3457 853 plusmn 27451 643 plusmn 11419 467 plusmn 16486 614 plusmn 55447lowastpH measured in CaCl2 001mol Lminus1 Ca2+ Mg2+ and Al3+ extracted with KCl 1mol Lminus1 H+ + Al3+ extracted with calcium acetate 05mol Lminus1 K+ and Pextracted with the Mehlich-1 extractor C determined with K2Cr2O7

Therefore the objective of this experimentwas to evaluateAMF colonization and spore AMF richness in nursery soilsamples at the metropolitan region of Curitiba Brazil withthe interest of identifying the most abundant taxonomicalAMF groups that grow under those conditions

2 Material and MethodsAraucaria seedlings were produced from seeds in the nurseryof the Universidade Federal do Parana Curitiba (25∘2510158404710158401015840Sand 49∘1610158401910158401015840W 950m) Brazil under entirely randomdesign five replicates and five treatmentsThe substrate usedwas a mixture of soil and organic residues (crop residuesorganic compounds and others) collected in the metropoli-tan area of the city The experimental design was completelyrandomized with five replications in each treatment Theseedlings were at the moment of harvesting according tothe respective treatments 6-month 1-year 2-year and 5-year old Seedlings grew outdoors under full light naturalconditions and were well watered during the whole period ofexperiment The climate in the region is classified as KoppenCfb [15] and the eventual climate events such as frost andstorms did not damage any stage of plant growth

Soil samples were submitted to chemical analyses andthe pH was measured in CaCl2 001mol Lminus1 Ca2+ Mg2+and Al3+ were extracted with KCl 1M H+ + Al3+ withCa(C2H3O2)2 05M K+ and P with the Mehlich-1 extractorcarbon (C) with the K2Cr2O7 (Table 1)

Mycorrhizal spores were extracted with minor modifica-tions with the procedures of [16 17] as follows 100 g of soilsample was suspended in 100ml and left resting for 60minsoil suspensionswere liquefied for 10 s and sequentially sievedthrough 500 and 53 120583m mesh sieves spores were recoveredin tubes with 70 sucrose solution and centrifuged for fourmin twice and then sieved for 250 and 50120583m mesh sievesSpores with similar morphology were assembled then theywere spread on microscope slide and treated with polyvinylalcohol-lactic acid-glycerol [18] and the Melzer reagent [19]Spores were individually identified undermicroscope (PrimoStarZeiss) according to the morphological characteristicsusing the criteria of taxonomical classification proposed byInvam [20]

Root fragments were preserved in lactoglycerol (lacticacid glycerol and water in the proportion 1 2 1) before

100

80

60

40

20

0

Root

colo

niza

tion

()

c

ab abba

6 months 1 year 2 years 3 years 5 yearsAraucaria seedling age (year)

Figure 1 Root arbuscular mycorrhizal fungi (AMF) colonizationaraucaria seedlings with ages between 6 months and 5 years grownin a nursery in field in Curitiba Brazil Means followed by the sameletter are not significantly different according to Tukeyrsquos test (119901 le005)The error bars represent the standard error of themean 119899 = 5

measurements The rates of mycorrhizal colonization wereestimated by observing cleared and dyed root fragmentsunder microscope (Stemi 305Zeiss) considering a grid of1 cm times 1 cm [21] with minor modifications as follows fineroots were soaked in KOH 10 at room temperature during24 hours and then in water bath at 80∘C for an hour rootswere sequentially washed with H2O2 100 and water twiceand dyed with blue-pen ink in water bath at 80∘C for fiveminutes

Variance analyses and mean comparisons considered thestatistical design of entirely random design five replicatesand five treatments Redundancy analyses were performedwith Canoco for Windows 45 package 43 considering theabundance of spore taxonomic group and chemical attributesas the explanatory variables for the differences between thetreatments

3 Results

Araucaria seedlings with ages between 6 months and 5 yearsproduced under nursery conditions in Curitiba were wellcolonized by AMF (rates varying from almost 50 to over 85Figure 1) and contained an abundant number of mycorrhizal









Glomus sp 2 91a 142a 55a 161a 141a


Glomus sp 4 48b 153a 57b 73b 148a







4 Discussion








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5 Conclusions





Acknowledgments




References








































Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy












Glomus sp 2 91a 142a 55a 161a 141a


Glomus sp 4 48b 153a 57b 73b 148a







4 Discussion








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5 Conclusions





Acknowledgments




References








































Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy






063 minus018

minus043 031

minus072 014

018 minus052

minus008 minus040

064 minus014

minus007 minus055

minus044 minus025



10

10

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Ca (cmol＞Ｇ3)

Mg (cmol＞Ｇ3)

K (cmol＞Ｇ3)

pH Ｆ2

C (g＞Ｇ3)





5 Conclusions





Acknowledgments




References








































Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy






References








































Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy












Journal of






Microbiology



Volume 2018



Volume 2018















BotanyJournal of










Agronomy




colonization and spore richness of arbuscular mycorrhizal...

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