Pisolithus Eucalyptus - Hindawi Publishing Corporationdownloads.hindawi.com/journals/aess/2015/803821.pdfEucalyptus seedlings were inoculated with cultures of Pisolithus previously

Research ArticleAluminum-Tolerant Pisolithus Ectomycorrhizas ConferIncreased Growth Mineral Nutrition and Metal Tolerance toEucalyptus in Acidic Mine Spoil

Louise Egerton-Warburton12

1Chicago Botanic Garden 1000 Lake Cook Road Glencoe IL 60022 USA2Program in Plant Biology and Conservation Weinberg College of Arts and Sciences Northwestern University EvanstonIL 60208 USA

Correspondence should be addressed to Louise Egerton-Warburton lwarburtonchicagobotanicorg

Received 9 April 2015 Revised 14 August 2015 Accepted 27 August 2015

Academic Editor Oliver Dilly

Copyright copy 2015 Louise Egerton-Warburton 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

Ectomycorrhizal fungi (ECM) may increase the tolerance of their host plants to Al toxicity by immobilizing Al in fungal tissuesandor improving plant mineral nutrition Although these benefits have been demonstrated in in vitro (pure culture) or short-term nutrient solution (hydroponic) experiments fewer studies have examined these benefits in the field This study examinedthe growth mineral nutrition and Al levels in two Eucalyptus species inoculated with three Pisolithus ecotypes that varied in Altolerance (in vitro) and grown inmine spoil in the greenhouse and field All three ecotypes of Pisolithus improvedEucalyptus growthand increased host plant tolerance to Al in comparison to noninoculated plants However large variations in plant growth andmineral nutrition were detected among the Pisolithus-inoculated plants these differences were largely explained by the functionalproperties of the Pisolithus inoculum Seedlings inoculated with the most Al-tolerant Pisolithus inoculum showed significantlyhigher levels of N P Ca Mg and K and lower levels of Al than seedlings inoculated with Al-sensitive ecotypes of Pisolithus Thesefindings indicate an agreement between the fungal tolerance to Al in vitro and performance in symbiosis indicating that bothECM-mediated mineral nutrient acquisition and Al accumulation are important in increasing the host plant Al tolerance

1 Introduction

Potentially toxic levels of aluminum (Al3+) are a major con-straint to plant growth during the restoration of acidicminingsoils Exposure to Al generally results in a severe reductionin growth and productivity due to the inhibition of root celldivision and elongation and reductions in the uptake of waterand assimilation of nutrients including N P Ca and MgCertain plant families show an innate tolerance to acidic soilsandAl [1] and some trees species are thought to resist elevatedsoil metal concentrations by means of a large phenotypicplasticity [2] For many tree species however symbioses witha small guild of well-adapted ectomycorrhizal (ECM) fungireduce the sensitivity of roots and plants to Al stress [3]

Many trees species form symbiotic associations withECM fungi These fungi provide nutrients and water to trees

in exchange for carbohydrates and may also play a crucialrole for tree regeneration in soils contaminated with metalsIn vitro studies indicate that ECM fungi may tolerate high Alconcentrations [4 5] and show wide inter- and intraspecificvariations in sensitivity to Al and other phytotoxic metals[6 7] For example studies showed that Laccaria was moretolerant of high Al availability than Hebeloma or Lactarius[4 8] and Pisolithus and Suillus were more tolerant of AlthanThelephora [5] In addition tolerance may be correlatedwith soil metal concentrations at the site of origin [9] but notalways [5]

In symbiosis ECM fungi may show broad ameliorativeeffects on woody plant responses to acidification [10] andphytotoxic metals including Al Cu Ni and Zn [6 11] Asa result plants inoculated with ECM generally maintainbetter growth and reduced transfer of metals to shoots than

Hindawi Publishing CorporationApplied and Environmental Soil ScienceVolume 2015 Article ID 803821 9 pageshttpdxdoiorg1011552015803821

2 Applied and Environmental Soil Science

Table 1 The percentage of soil separates and levels of pH nutrients and Al in soil and mining spoil from each of the Pisolithus sporocarpcollection sites Values represent the mean with standard error in parentheses For each column means with the same letter do not differsignificantly at p lt 005

SiteSand Silt Clay

pHN C P Al Mn Ca Mg K

120583g gminus1 soil Meq 100 gminus1 soil

Mine 58 21 21 460b(006)

0029b(001)

571b(15)

068c(023)

320a(45)

631a(105) 012 051 003

Restored 81 6 13 479b(011)

0034b(001)

1023a(12)

266a(034)

36b(5)

298b(129) 009 040 001

Forest 73 8 19 561a(026)

0131a(001)

545b(13)

178b(023)

10c(3)

156b(99) 600 410 030

nonmycorrhizal plants when exposed to acidity andor anexcess availability of Al [11] Such ECM-mediated metal tol-erance has been attributed to various extracellular (chelationcell-wall binding) or intracellular detoxification mechanismssuch as binding to nonprotein thiols or vacuolar storage[1 4 11ndash14] In other studies however the alleviation of Alor heavy metal toxicity by ECM was attributed to improvedplant mineral nutrition owing to an increase in the uptakeof poorly soluble ions (P) or base cations (Ca Mg) [4 1015 16] Further the effects of ECM on plant Al tolerancemay depend on the identity of the host species or ECM-host combination For example inoculation with Laccariaconferred Al tolerance to both Fagus and Pinus whereasinoculation with Paxillus conferred tolerance only to Pinus[10] In acidic soils Xerocomus badius-Picea abies ECMshowed a higher potential to store N K Mg and metalsin the fungal sheath than other ECM-Picea combinations[12]

Despite this increasing knowledge base there are stillsurprisingly few field experiments that test for relevantdifferences in Al tolerance in ECM-host tree combination [417] Insteadmost of the reported effects of ECMonAl toxicityand plant growth have been generated in short-term nutrientsolution (hydroponic) experiments [8] and these results donot always accurately predict plant field performance [18]This study examined the growth andmineral nutrition of twoEucalyptus species inoculated with three Pisolithus ecotypeswhen grown in acid mine spoil in the greenhouse and fieldThe Pisolithus ecotypes were collected from coal mine spoilthat differed in soil Al levels and were previously found toshow varying levels of adaptive tolerance to Al [9] Pisolithusis a cosmopolitan genus that forms ECM associations witha broad range of angiosperm and gymnosperm tree speciesincluding Eucalyptus [19] It is also a common pioneer onmine sites and increases plant growth in adverse soil condi-tions [17] The results were used to address three questions(1) Do Pisolithus isolates from soils with the highest Al levelsprovide the greatest benefits to seedlings growing in minespoil (2) Does the effect of the Pisolithus isolates on plantgrowth vary with host plant identity (3) If there is an effectofPisolithus isolates on seedling growth can this be attributedto reducedAl accumulation in plant tissues or improved plantnutrition

2 Materials and Methods

Seed inoculum and soil for the pot trial were all sourcedfrom the lease of Western Collieries Ltd which is located inthe Collie Coal Basin Western Australia (33∘261015840S 116∘121015840E)The Collie Coal Basin occurs in a significant physiognomicdepression of the Darling Plateau in southwestern AustraliaThe region is typified by a warmMediterranean-type climateand receives on average 993mm precipitation per annumRainfall occurs fromMay to October (Southern Hemispherewinter-spring) followed by five to six months with little orno rainfall (Southern Hemisphere summer-autumn) Forestsoils are typically nutrient poor leached sands (Table 1)In contrast coal-mining soils are characterized by waterrepellence and composed of coarse nonbound sands withsignificant increases in the proportion of silt and clay incomparison to forest soils (Table 1) Mine spoil is also moreacidic and contains higher levels of Al and Mn and lowerlevels of N P Ca Mg and K than forest soils (Table 1)

21 Pisolithus Inoculum Preparation Eucalyptus seedlingswere inoculated with cultures of Pisolithus previously iden-tified as varying in Al tolerance [9] Briefly Pisolithus wasisolated from sporocarps collected under Eucalyptus trees inabandoned mine sites restored sites and native forest sites(Table 1) and sequentially cultured to produce pure cultureIn vitro screening in Al-amended solid media identifiedvariations in Al tolerance among the Pisolithus isolateswhereby Pisolithus that was isolated from abandoned andrestored mine sites was highly tolerant of Al (as Al3+) andPisolithus isolated from an adjacent native forest showedsignificantly lower tolerance to Al [9] Intersimple sequencerepeat (ISSR) fingerprinting also revealed strong intraspe-cific variation among these Pisolithus isolates (Egerton-Warburton unpublished data) These isolates were grown inbulk and then encapsulated in hydrogel beads [20] as thiscomprised an efficient method for the large-scale inoculationof containerized seedlings

22 Greenhouse Trial Two common forest trees used inrestoration Eucalyptus rudis Endl and Eucalyptus patensBenth were used in this study Seeds of each species werecollected frommature trees growing in abandonedmine siteson the lease of Western Collieries Ltd at Collie Half-sibling

Applied and Environmental Soil Science 3

progeny was collected from an individual tree to reduce theeffect of genetic variation within the seed stock on the trialSeeds were surface sterilized with 3 vv NaHClO

4for 5min

washed in three changes of sterile deionized water and thentransferred to moistened sterile filter paper in petri dishesSeedswere incubated in darkness at room temperature (20∘C)for eight days before transplanting into prepared pots ofminespoil

Mine spoil was used as the growing medium Thesematerials were classified as a sandy loam (738 sand 162clay 101 silt) acidic (pH 446 plusmn 04 mean plusmn se) with highlevels of Al (327 plusmn 45 120583g gminus1 soil) and low levels of plant-available P (024 plusmn 002 120583g gminus1 soil) and available moisture(68ndash11) Soils were sieved to 2mm and then 15 kg wasplaced in each of 240 plastic pots lined with polythenebags that had been perforated at the base to allow drainageForty replicate pots per inoculum source plus 40 replicatepots of noninoculated pots (control) were established foreach Eucalyptus species For each species and inoculumsource four germinants were placed in an individual pot andsupplied with eight hydrogel beads (two per plant) in theroot zone After planting N and P fertilizers were appliedonce in solution to the soil surface at rates of 2mg P kgminus1 soil(as KH

2PO4) and 2mgNkgminus1 soil (as NH

4NO3) No further

fertilizers were applied during the trial Soils were wateredto 8 soil moisture content and maintained in a greenhouse(30∘C max 21∘C min) Pots were segregated by inoculumsource to reduce the possibility of cross-contamination Atthe four-leaf stage seedlings were thinned to one per potand any cotyledons were removed from seedlings to promotemycorrhization Soil moisture levels were maintained bybringing pots to field capacity twice a week

Seedlings were destructively harvested after 150 daysof growth At harvest plants were washed gently frompots and divided into roots and shoots Root material wassubdivided into coarse and fine roots with subsamples offine roots from each treatment and species inspected forthe abundance color and morphology of the colonisingmycorrhizas Short roots with fully developed sulfur yellowmantles and emanating hyphae were considered mycorrhizasof Pisolithus Mycorrhizal colonization was expressed as thepercentage of lateral fine roots colonizedThe remaining rootand shoot materials were dried to constant weight at 60∘Cand dry weights recorded Subsamples of shoot material ofeach Eucalyptus and inoculum treatment were then groundto a fine powder and analyzed for N P K Ca Mg and Al byCSBP and Farmers Analytical Laboratory (Bayswater West-ern Australia httpwwwcsbp-fertiliserscomaucsbp-lab)

23 Field Trial Seedlings of E rudis and E patens wereinoculated with mine or forest site isolates of Pisolithus andassessed for growth andmycorrhization in the field Seedlingsinoculated with Pisolithus from the restored sites were notincluded in this comparison since the growth responses andnutrient levels were largely similar to those in seedlingsgrown with mine site inoculum (see Figure 1) In additionnoninoculated plants were not included in this assessmentas their survival was extremely poor (lt15) in pot trials

Seeds were germinated as described previously For eachspecies and inoculum source a single germinant was placedin an individual Jiffy pots (capacity 50mL) containing sievedCollie topsoil which is classed as sand (93 sand 24 clay46 silt) with pH 53 extractable Al 2120583g gminus1 soil and P16 120583g gminus1 soil Seedlings were supplied with hydrogel bead(two per plant) in the root zone Supplemental fertilizationof forest soil was not required owing to the higher levelsof plant-available P Sixty-four seedlings of each species andinoculum typewere established (total = 256 plants) Seedlingswere maintained in a shade house and watered daily with anautomatic watering system for four months prior to planting

Seedlings were planted into a prepared rehabilitation siteon the lease of Western Collieries Ltd at Collie The sitehad been prepared by battering the subsoil to a 9∘ slopefollowed by a dressing of local forest topsoil and a fertilizerapplication of 10 kg P haminus1 as single superphosphate (9 P)Eighteen months after planting the surviving seedlings wereexcavated from the field site and processed for mycorrhizalcolonization root and shoot biomass and mineral nutrientcontent as described previously (see Section 22 GreenhouseTrial)

24 Data Analyses The differences in total plant biomass(root shoot) and foliar mineral nutrient levels (pot fieldtrials) and mycorrhizal colonization (pot trial) were ana-lyzed using analyzed linear mixed models with restrictedmaximum likelihood (REML) estimates In the analysesEucalyptus host species and Pisolithus inoculation source(mine restored and forest) were specified as random effectsBecause both host plant and mycorrhizal partner can havelarge influences on host nutrition the relative contributionof Eucalyptus host species and Pisolithus inoculum sourceon plant mineral nutrition was tested using REML linearmodels Biomass accumulation was modeled as a function ofthe fixed effect of plant nutrient levels (N P K Ca Mg andAl) with Eucalyptus host and Pisolithus source specified asrandom effects To determine the combinations of nutrientswith the greatest explanatory power each variable (mineralnutrient) was added into the model in a stepwise fashionBriefly variables that were found to be significant in thepresence of previously fitted variables were retained in themodel while variables that were no longer significant in thepresence of other variables were removedThefinal combinedmodels were used to report the variance explained by planthost and inoculum source in the pot and field trials In addi-tion variables retained in the model were tested using posthoc multiple 119905-tests to determine significant differences inmineral nutrients between plant hosts or inoculum sourcesAll variables were tested for normality and where necessarylog transformations were applied prior to analysis

3 Results

31 Greenhouse Trial Eucalyptus host (119901 lt 0001) influencedplant root and shoot biomass accumulation whereby Erudis plants were significantly larger than E patens plants(Figure 1(b)) Shoot biomass in E patens and E rudiswas sig-nificantly higher (119901 lt 0001 Figure 1(a)) in inoculated than


MineRestored

ForestControl

0

200

400

600

800

1000

1200

E patens E rudis

AA A

A AA

C B

Shoo

t mas

s (m

gmiddotpl

antminus1

)

(a)

E patens E rudis

A

A A A

B

B

C

D0

200

400

600

800

1000

1200

Shoo

t mas

s (m

gmiddotpl

antminus1

)

MineRestored

ForestControl

(b)

0

20

40

60

80

100

E patens E rudis

ECM

root

tips

()

A

B

C

B

C

A

B B

MineRestored

ForestControl

(c)

Figure 1 Effect of mine restored or forest site Pisolithus ecotype on the shoot biomass (a) root biomass (b) and fine root ectomycorrhizalcolonization (c) in Eucalyptus plants in the pot trial Vertical bars indicate the standard error of the mean For each plant species meanbiomass or root colonization values with the same letter do not differ significantly at 119901 lt 005

in noninoculated (control) plants but there was no differencein shoot biomass among Pisolithus sources (119901 = 0274) Rootbiomass in E patens and E rudiswas also significantly higher(119901 lt 0001 Figure 1(b)) in inoculated than in noninocu-lated (control) plants In E patens there was no significantdifference in root biomass among Pisolithus sources (119901 =0267) whereas E rudis inoculated with Pisolithus fromminesites produced significantly greater root biomass than wheninoculated with Pisolithus from restored or forest sites (119901 =0002)

Both Eucalyptus host (119901 lt 0001) and Pisolithus source(119901 lt 0001) influenced mycorrhizal root colonization(Figure 1(c)) Root colonization was significantly higher inE rudis (54 plusmn 6 mean plusmn se) than E patens (26 plusmn 3)In addition 61 plusmn 9 root tips were colonized in plantsinoculated with mine site Pisolithus compared with 41 plusmn 6for restored site and 43 plusmn 5 for forest site Pisolithus There

was no significant Eucalyptus x Pisolithus source interaction(119901 = 0401)

Results from REML (Table 2) revealed that variations inplant N P and Ca (positive) andAl content (negative) signifi-cantly influenced biomass accumulation and such variationswere largely explained (684) by the functional propertiesof the Pisolithus inoculum Seedlings inoculated withmine orrestored site Pisolithus inoculum showed significantly higherlevels of N P and Ca and lower levels of Al than seedlingsinoculated with forest site Pisolithus (119901 lt 0001 Figures2(a) 2(b) 2(d) and 2(f)) Noninoculated seedlings containedthe lowest levels of mineral nutrients and highest levels ofAl Levels of K and Mg were also significantly higher inseedlings inoculatedwithmine or restored site than forest sitePisolithus (119901 lt 0001 Figures 2(c) and 2(e) resp) but thesenutrients were not significant in REML models of biomassaccumulation


Table 2 Residual maximum likelihood (REML) results of the influence of Eucalyptus species and Pisolithus isolate source as explanatoryfactors on plant biomass and mineral nutrition in pot and field trials The significant fixed effects (nutrients) their standardized estimateswith standard error (se) p values and the amount of variation explained by plant and mycorrhizal parameters are presented

Response variable Retained fixed effect Estimate (se) p Eucalyptus species () Pisolithus source ()

Biomass (pot)

N 0164 (009) 0004

269 684P 1075 (003) lt0001Ca 0858 (022) 0010Al minus0518 (007) 0008

Biomass (field)

N 0726 (008) 0004

271 586P 1990 (021) 0007Ca 0226 (002) 0005Mg 0275 (006) 0008Al minus0477 (004) 0002

32 Field Trial Eucalyptus host (119901 = 0003) and Pisolithussource (119901 = 0006) influenced plant biomass accumulationafter 18 months in the field (Figure 3(a)) There was nosignificant Eucalyptus x Pisolithus source interaction (119901 =0513) Eucalyptus rudis was significantly larger than Epatens and Pisolithus frommine sites resulted in greater plantbiomass than Pisolithus from the forest site

Overall levels of ECM root colonization did not differsignificantly between seedlings receiving mine site (74 plusmn4 mean plusmn se) or forest sources of Pisolithus (77 plusmn 3)However five different morphological types of ECM includ-ing Pisolithus colonized root tips Pisolithus was readilyidentified by the presence of a coarse sulfur yellow mantleemanating hyphae and rhizomorphs One ECM was con-sistent with Cenococcum (black mantle short black hyphae)while the other was an unknown type characterized by adeep brown mantle and short radiating external hyphae Infact after 18 months of field growth Pisolithus mycorrhizascomprised on average only 19 plusmn 4 of ECM root tips inseedlings receiving mine site inoculum and 7 plusmn 3 of ECMroot tips in seedlings receiving forest inoculum

Results from REML analyses for field-grown plants weresimilar to those from the pot trial variations in plant N P CaandMg (positive) and Al (negative) influenced plant biomassaccumulation (Table 2) In addition these variations werebetter explained by the properties of the Pisolithus inoculum(586) than host plant Plant N levels were significantlyhigher in seedlings inoculated with mine (1199 plusmn 18mgNplantminus1 meanplusmn se) than forest sitePisolithus (1176 plusmn 7mgNplantminus1 119901 = 0018) In addition levels of plant K (119901 lt 0001)Ca (119901 lt 0001) Mg (119901 lt 0001) and P (119901 = 0009) weresignificantly higher and Al levels were significantly lower(119901 lt 0001) in plants inoculated with mine than forest sitePisolithus (Figure 3(b))

4 Discussion

An extensive literature has shown that ECM fungi frommetal-contaminated soils may present a higher metal tol-erance (in vitro) than isolates from noncontaminated soilsand that intraspecific variation in metal tolerance can occuramong isolates [3 6 7 9] However questions have remained

about whether the adaptive metal tolerance observed in vitromight result in increased protection against metal toxicityin a host plant growing in contaminated soils In this studyall three ecotypes of Pisolithus improved Eucalyptus growthand mineral nutrition (N P Ca Mg and K) and reducedthe transfer to a phytotoxic metal (Al) to the host plant incomparison to nonmycorrhizal seedlings These trends arein general agreement with earlier studies of ECM plants inmetal-contaminated and native soils [3 11 15] More notablythe experiments revealed that the source of the Pisolithusisolate was the largest influence of plant growth mineralnutrition and Al content

Overall Eucalyptus seedlings inoculated with minePisolithus produced the largest growth responses and forestPisolithus the least The magnitude of this effect is mostobvious in E rudis (pot trial) and in shoot biomass accu-mulation in the field trial (both Eucalyptus species) Becausethe study used half-sib progeny and confined plant growthto a single substrate (mine spoil) these results suggest thatgenetic differences existed between Pisolithus isolates in theirability to benefit seedling growth [3 6 21] In addition plantgrowth responses were largely consistent with the patternsof Pisolithus Al tolerance reported in in vitro experiments[9] suggesting a reasonable agreement between the fungalsensitivity to specific metals in vitro and performance insymbiosis in acidic mine spoil (Question 1)

Even so there were large variations in the symbiotic andphysiological effectiveness of Pisolithus between host speciesE rudis showed high levels of mycorrhizal root colonizationand correspondingly larger growth responses to Pisolithusin comparison to E patens (Question 2) This result is notsurprising given the wide range of physiological responsesthat have been reported in host species inoculated withPisolithus [22 23] What was surprising however was thatall three isolates of Pisolithus produced similar root andshoot growth responses in E patens (pot trial) While itis possible this result represents some form of fungal-hostincompatibility between Pisolithus and E patens [21 23] themore likely explanation is that Pisolithus acted as a strong Csink during the early stages of the symbiosis [24] Eucalyptuspatens grownwithmine sitePisolithus for 4months (pot trial)showed the highest levels of root colonization but limited


AAB

C

N co

nten

t (m

gmiddotpl

antminus1

)

0

2000

4000

6000

8000

Mine Restored Forest Control(a)

C

AA

AB

0

100

200

300

400

500

600

P co

nten

t (m

gmiddotpl

antminus1

)

Mine Restored Forest Control(b)

C

AAB

B

0

1000

2000

3000

K co

nten

t (m

gmiddotpl

antminus1

)

Mine Restored Forest Control(c)

C

A A

B

0

1000

2000

3000Ca

cont

ent (

mgmiddot

plan

tminus1

)

Mine Restored Forest Control(d)

Inoculum source

C

A

AB

B

0

500

1000

1500

2000

2500

Mg

cont

ent (

mgmiddot

plan

tminus1

)

Mine Restored Forest Control

(e)

C

A

BB

Inoculum sourceMine Restored Forest Control

200

300

400

500

600

Al c

onte

nt (m

gmiddotpl

antminus1

)

(f)

Figure 2 Effect of inoculation with mine restored or forest site Pisolithus ecotype on the mean foliar N (a) P (b) K (c) Ca (d) Mg (e) andAl levels (f) in Eucalyptus plants in the pot trial Vertical bars indicate the standard error of the mean For each nutrient mean levels with thesame letter do not differ significantly at 119901 lt 005


E patens E rudis

MineForest

Host plant

Shoo

t mas

s (m

gmiddotpl

antminus1

)

lowast

lowast

0

100

200

300

400

500

600

(a)

K Ca Mg Al P

MineForest

Mineral nutrient

lowast

lowast

lowast

lowast

lowast

0

100

200

300

400

500

Nut

rient

cont

ent (

mgmiddot

plan

tminus1

)

(b)

Figure 3 Effect of inoculation with mine or forest site Pisolithus ecotype on shoot biomass (a) and foliar levels of K Ca Mg Al and P (b)in Eucalyptus plants in the field trial Vertical bars indicate the standard error of the mean For each plant species (a) or nutrient (b) meanvalues indicated with an asterisk (lowast) differ significantly at 119901 lt 005 ns not significantly different (119901 gt 005)

biomass accumulation After 18 months of field growthhowever root colonization and seedling biomass in E patenswere similar to E rudis These results support the idea thatearly symbiosis may have acted as a temporary C sink in Epatens

At least two general types of mechanisms apparentlycontributed to the enhanced growth response in E rudiswith mine site Pisolithus (Question 3) The most obviouswas the ability of the ECM partner to reduce Al levelswithin the plant In plants grown with mine site PisolithusAl levels remained low in comparison to plants grown withforest Pisolithus These experiments were not intended toevaluate the specific mechanisms by which ECM fungi mightalleviate Al toxicity However the significant differencesin foliar Al levels between nonmycorrhizal and Pisolithus-inoculated plants indicate that mycorrhizal structures mayhave limited Al penetration into the root symplasm andsubsequent transport to the shoot [11 25] Binding of Al tothe fungal cell walls represented a substantial fraction of themetal accumulated by Pisolithus mycorrhizas on Eucalyptus[9] and Pinus [26] and may well be part of the mechanismby which ECM Eucalyptus tolerated the high levels of themetal in spoil in the current study In addition studieswith Pisolithus and other ECM fungi have highlighted a rolefor Al detoxification within the fungal vacuole with S-richsubstrates or P-rich granules [12ndash14 27] and the capacity ofPisolithus mycelia to produce low molecular-weight organicacids that bind Al and prevent its absorption [4 25 28]Although it is uncertain which processes might be involvedthe reduction in Al levels in ECM Eucalyptus in this studyimplies an efficient and substantial tolerance mechanism

A second type of tolerance mechanism appeared to bethemaintenance of nutrient acquisitionMycorrhizal E rudiscontained significantly higher levels of N P K Ca and Mgthan nonmycorrhizal plants inmine spoilThis is particularlyimportant because mine spoil is largely deficient in essential

plant nutrients such as N P and K and high levels of Alare known to limit the uptake of divalent cations (Ca Mg)Indeed the stunted root growth and low levels of foliarN P K Ca and Mg in nonmycorrhizal E rudis and Epatens were consistent with Al-induced growth impairmentsin woody plants [25] and competitive inhibition of Mg andCa uptake [16 29] In contrast plants grown with Pisolithusshowed substantial increases in N and P consistent with thephysiological role of ECM in natural forest or Al-treatedsoils [4 10 25] as well as increased plant Mg Ca and Klevels This result suggests that an ECM-mediated uptake ofcations might directly ameliorate the effects of Al [4 8 16]in a manner similar to other phytotoxic metals [11 15] orindirectly by enhancing P uptake [30] Taken together withplant Al levels these findings suggest that both plant nutrientimbalances and direct Al toxicity are likely the main reasonsfor the negative effects of mine spoil on plant growth

It is interesting to note that inoculation with mine sitePisolithus resulted in the greatest increases in plant growthand nutrient status and forest Pisolithus resulted in thesmallest increases This outcome agrees with earlier findingsshowing the high intraspecific variability of Pisolithus (andother ECM fungi) to promote the mineral nutrition of thehost plant [21 22 31] However other key factor(s) besidesnutrient acquisition may be involved Pisolithus produceslong distance exploration hyphae with hydrophobic cell wallsthat are capable of transporting physiologically significantquantities of water through the soil-plant continuum [32]This is notable because mine spoil shows low water holdingcapacity and juvenile Eucalyptus growth is strongly and neg-atively influenced by soil moisture deficits The inoculationof woody plants with Pisolithus has been shown to mitigatethe effects of drought by improving water acquisition ormodifying plant ecophysiological responses to soil moisturedeficits [31] Because genotypes of Pisolithus differ markedlyin their capacity to confer drought tolerance to their host [31]


it is tempting to speculate that Pisolithus and especially themine site isolate may have enhanced the growth of E rudisby ameliorating the effects of soil moisture deficits Given thepotential importance of this mechanism further field-basedexperimentation is needed to determine the impact of thedifferent Pisolithus isolates on Eucalyptus ecophysiology

5 Conclusions

InoculationwithPisolithus appeared to compensate for ineffi-cient plant nutrient acquisition as well as bestow increased Alresistance in a host plant that showed a level of sensitivity tospoil conditions Pisolithus isolates also differed markedly intheir capacity to confer such benefits to their host indicatingthat screening and selecting fungal isolates based on theircapacity to adapt to abiotic stresses in the spoil such as Almay be a prerequisite to using ECM isolates for larger scaleinoculation trials or restoration Although the potential existsto utilize the most Al-tolerant ECM it was also clear thatnot all Eucalyptus species would benefit from inoculationduring early seedling establishment As a result screeningthe performance of various Pisolithus-Eucalyptus speciescombinations in mine spoil comprises a logical second stepin planning for future field planting

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by a Commonwealth ForestryPostgraduate Award andWestern Collieries Ltd (WCL) Theauthor thanks Bob Brierley (WCL) for assistance with siteselection and seedling outplanting and Dr Clem Kuek forcreating the hydrogel beads used for inoculation

References

[1] I Brunner and C Sperisen ldquoAluminum exclusion and alu-minum tolerance in woody plantsrdquo Frontiers in Plant Sciencevol 4 article 172 2013

[2] D M Wilkinson and N M Dickinson ldquoMetal resistance intrees the role ofmycorrhizaerdquoOikos vol 72 no 2 pp 298ndash3001995

[3] J V Colpaert J H L Wevers E Krznaric and K AdriaensenldquoHow metal-tolerant ecotypes of ectomycorrhizal fungi protectplants fromheavymetal pollutionrdquoAnnals of Forest Science vol68 no 1 pp 17ndash24 2011

[4] U Ahonen-Jonnarth A Goransson and R D Finlay ldquoGrowthand nutrient uptake of ectomycorrhizal Pinus sylvestris seed-lings in a natural substrate treated with elevated Al concentra-tionsrdquo Tree Physiology vol 23 no 3 pp 157ndash167 2003

[5] G W Thompson and R J Medve ldquoEffects of aluminum andmanganese on the growth of ectomycorrhizal fungirdquo Appliedand Environmental Microbiology vol 48 pp 556ndash560 1984

[6] J Hartley J W G Cairney and A A Meharg ldquoDo ectomy-corrhizal fungi exhibit adaptive tolerance to potentially toxic

metals in the environmentrdquo Plant and Soil vol 189 no 2 pp303ndash319 1997

[7] D Blaudez C Jacob K Turnau et al ldquoDifferential responsesof ectomycorrhizal fungi to heavy metals in vitrordquoMycologicalResearch vol 104 no 11 pp 1366ndash1371 2000

[8] R H Jongbloed and G W F H Borst-Pauwels ldquoEffects ofaluminium and pH on growth and potassium uptake by threeectomycorrhizal fungi in liquid culturerdquo Plant and Soil vol 140no 2 pp 157ndash165 1992

[9] L M Egerton-Warburton and B J Griffin ldquoDifferential re-sponses of Pisolithus tinctorius isolates to aluminum in vitrordquoCanadian Journal of Botany vol 73 no 8 pp 1229ndash1233 1995

[10] R Finlay ldquoInteractions between soil acidification plant growthand nutrient uptake in ectomycorrhizal associations of foresttreesrdquo Ecological Bulletins vol 44 pp 197ndash214 1995

[11] G Jentschke andD L Godbold ldquoMetal toxicity and ectomycor-rhizasrdquo Physiologia Plantarum vol 109 no 2 pp 107ndash116 2000

[12] I Kottke X M Qian K Pritsch I Haug and F OberwinklerldquoXerocomus badiusmdashPicea abies an ectomycorrhiza of highactivity and element storage capacity in acidic soilrdquoMycorrhizavol 7 no 5 pp 267ndash275 1998

[13] H Bucking S BeckmannW Heyser and I Kottke ldquoElementalcontents in vacuolar granules of ectomycorrhizal fungi mea-sured by EELS and EDXS A comparison of different methodsand preparation techniquesrdquo Micron vol 29 no 1 pp 53ndash611998

[14] F Martin P Rubini R Cote and I Kottke ldquoAluminium pol-yphosphate complexes in the mycorrhizal basidiomycete Lac-caria bicolor a 27Al-nuclear magnetic resonance studyrdquo Plantavol 194 no 2 pp 241ndash246 1994

[15] P Jourand L Hannibal C Majorel S Mengant M Ducoussoand M Lebrun ldquoEctomycorrhizal Pisolithus albus inoculationof Acacia spirorbis and Eucalyptus globulus grown in ultramafictopsoil enhances plant growth and mineral nutrition whilelimits metal uptakerdquo Journal of Plant Physiology vol 171 no 2pp 164ndash172 2014

[16] J Bose O Babourina and Z Rengel ldquoRole of magnesium inalleviation of aluminium toxicity in plantsrdquo Journal of Experi-mental Botany vol 62 no 7 pp 2251ndash2264 2011

[17] D H Marx and J D Artmann ldquoPisolithus tinctorius ectomyc-orrhizae improve survival and growth of pine seedlings in acidcoal spoils in Kentucky and Virginiardquo Reclamation Review vol2 pp 23ndash31 1979

[18] P H Nygaard andH A deWit ldquoEffects of elevated soil solutionAl concentrations on fine roots in amiddle-agedNorway spruce(Picea abies (L) Karst) standrdquo Plant and Soil vol 265 no 1-2pp 131ndash140 2004

[19] FMartin J Dıez B Dell andCDelaruelle ldquoPhylogeography ofthe ectomycorrhizal Pisolithus species as inferred from nuclearribosomal DNA ITS sequencesrdquoNew Phytologist vol 153 no 2pp 345ndash357 2002

[20] C Kuek I Tommerup and N Malajczuk ldquoHydrogel beadinocula for the production of ectomycorrhizal eucalypts forplantationsrdquo Mycological Research vol 96 no 4 pp 273ndash2771992

[21] T Burgess B Dell and NMalajczuk ldquoVariation inmycorrhizaldevelopment and growth stimulation by 20 Pisolithus isolatesinoculated on to Eucalyptus grandis WHill ex Maidenrdquo NewPhytologist vol 127 no 4 pp 731ndash739 1994

[22] T I Burgess N Malajczuk and T S Grove ldquoThe ability of16 ectomycorrhizal fungi to increase growth and phosphorus


uptake of Eucalyptus globulus Labill and E diversicolor FMuellrdquo Plant and Soil vol 153 no 2 pp 155ndash164 1993

[23] J W G Cairney ldquoIntraspecific physiological variation implica-tions for understanding functional diversity in ectomycorrhizalfungirdquoMycorrhiza vol 9 no 3 pp 125ndash135 1999

[24] JW Cairney A E Ashford andWG Allaway ldquoDistribution ofphotosynthetically fixed carbon within root systems of Eucalyp-tus pilularis plants ectomycorrhizal with Pisolithus tinctoriusrdquoNew Phytologist vol 112 no 4 pp 495ndash500 1989

[25] J R Cumming and L H Weinstein ldquoAluminum-mycorrhizalinteractions in the physiology of pitch pine seedlingsrdquo Plant andSoil vol 125 no 1 pp 7ndash18 1990

[26] K Moyer-Henry I Silva J Macfall et al ldquoAccumulation andlocalization of aluminium in root tips of loblolly pine seedlingsand the associated ectomycorrhiza Pisolithus tinctoriusrdquo PlantCell amp Environment vol 28 no 2 pp 111ndash120 2005

[27] K Turnau I Kottke and F Oberwinkler ldquoElement localizationinmycorrhizal roots ofPteridiumaquilinum (L) Kuhn collectedfrom experimental plots treated with cadmium dustrdquo NewPhytologist vol 123 no 2 pp 313ndash324 1993

[28] K TaharaMNorisada T TangeH Yagi andKKojima ldquoEcto-mycorrhizal association enhances Al tolerance by inducingcitrate secretion in Pinus densiflorardquo Soil Science and PlantNutrition vol 51 no 3 pp 397ndash403 2005

[29] L van Scholl W G Keltjens E Hoffland and N van BreemenldquoEffect of ectomycorrhizal colonization on the uptake of CaMg and Al by Pinus sylvestris under aluminium toxicityrdquo ForestEcology and Management vol 215 no 1ndash3 pp 352ndash360 2005

[30] H Bucking ldquoPhosphate absorption and efflux of three ectomy-corrhizal fungi as affected by external phosphate cation andcarbohydrate concentrationsrdquo Mycological Research vol 108no 6 pp 599ndash609 2004

[31] M S Lamhamedi P Y Bernier and J A Fortin ldquoGrowth nutri-tion and response to water stress of Pinus pinaster inoculatedwith ten dikaryotic strains of Pisolithus sprdquo Tree Physiology vol10 no 2 pp 153ndash167 1992

[32] M S Lamhamedi and J A Fortin ldquoGenetic variations of ecto-mycorrhizal fungi extramatrical phase of Pisolithus sprdquo Cana-dian Journal of Botany vol 69 no 9 pp 1927ndash1934 1991

Submit your manuscripts athttpwwwhindawicom

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Environmental and Public Health

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EcologyInternational Journal of


Marine BiologyJournal of


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Applied ampEnvironmentalSoil Science

Volume 2014

Advances in


Environmental Chemistry

Atmospheric SciencesInternational Journal of



Waste ManagementJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal of

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Geological ResearchJournal of

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BiodiversityInternational Journal of


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OceanographyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of Computational Environmental SciencesHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


ClimatologyJournal of


Table 1 The percentage of soil separates and levels of pH nutrients and Al in soil and mining spoil from each of the Pisolithus sporocarpcollection sites Values represent the mean with standard error in parentheses For each column means with the same letter do not differsignificantly at p lt 005

SiteSand Silt Clay

pHN C P Al Mn Ca Mg K

120583g gminus1 soil Meq 100 gminus1 soil

Mine 58 21 21 460b(006)

0029b(001)

571b(15)

068c(023)

320a(45)

631a(105) 012 051 003

Restored 81 6 13 479b(011)

0034b(001)

1023a(12)

266a(034)

36b(5)

298b(129) 009 040 001

Forest 73 8 19 561a(026)

0131a(001)

545b(13)

178b(023)

10c(3)

156b(99) 600 410 030

nonmycorrhizal plants when exposed to acidity andor anexcess availability of Al [11] Such ECM-mediated metal tol-erance has been attributed to various extracellular (chelationcell-wall binding) or intracellular detoxification mechanismssuch as binding to nonprotein thiols or vacuolar storage[1 4 11ndash14] In other studies however the alleviation of Alor heavy metal toxicity by ECM was attributed to improvedplant mineral nutrition owing to an increase in the uptakeof poorly soluble ions (P) or base cations (Ca Mg) [4 1015 16] Further the effects of ECM on plant Al tolerancemay depend on the identity of the host species or ECM-host combination For example inoculation with Laccariaconferred Al tolerance to both Fagus and Pinus whereasinoculation with Paxillus conferred tolerance only to Pinus[10] In acidic soils Xerocomus badius-Picea abies ECMshowed a higher potential to store N K Mg and metalsin the fungal sheath than other ECM-Picea combinations[12]

Despite this increasing knowledge base there are stillsurprisingly few field experiments that test for relevantdifferences in Al tolerance in ECM-host tree combination [417] Insteadmost of the reported effects of ECMonAl toxicityand plant growth have been generated in short-term nutrientsolution (hydroponic) experiments [8] and these results donot always accurately predict plant field performance [18]This study examined the growth andmineral nutrition of twoEucalyptus species inoculated with three Pisolithus ecotypeswhen grown in acid mine spoil in the greenhouse and fieldThe Pisolithus ecotypes were collected from coal mine spoilthat differed in soil Al levels and were previously found toshow varying levels of adaptive tolerance to Al [9] Pisolithusis a cosmopolitan genus that forms ECM associations witha broad range of angiosperm and gymnosperm tree speciesincluding Eucalyptus [19] It is also a common pioneer onmine sites and increases plant growth in adverse soil condi-tions [17] The results were used to address three questions(1) Do Pisolithus isolates from soils with the highest Al levelsprovide the greatest benefits to seedlings growing in minespoil (2) Does the effect of the Pisolithus isolates on plantgrowth vary with host plant identity (3) If there is an effectofPisolithus isolates on seedling growth can this be attributedto reducedAl accumulation in plant tissues or improved plantnutrition

2 Materials and Methods

Seed inoculum and soil for the pot trial were all sourcedfrom the lease of Western Collieries Ltd which is located inthe Collie Coal Basin Western Australia (33∘261015840S 116∘121015840E)The Collie Coal Basin occurs in a significant physiognomicdepression of the Darling Plateau in southwestern AustraliaThe region is typified by a warmMediterranean-type climateand receives on average 993mm precipitation per annumRainfall occurs fromMay to October (Southern Hemispherewinter-spring) followed by five to six months with little orno rainfall (Southern Hemisphere summer-autumn) Forestsoils are typically nutrient poor leached sands (Table 1)In contrast coal-mining soils are characterized by waterrepellence and composed of coarse nonbound sands withsignificant increases in the proportion of silt and clay incomparison to forest soils (Table 1) Mine spoil is also moreacidic and contains higher levels of Al and Mn and lowerlevels of N P Ca Mg and K than forest soils (Table 1)

21 Pisolithus Inoculum Preparation Eucalyptus seedlingswere inoculated with cultures of Pisolithus previously iden-tified as varying in Al tolerance [9] Briefly Pisolithus wasisolated from sporocarps collected under Eucalyptus trees inabandoned mine sites restored sites and native forest sites(Table 1) and sequentially cultured to produce pure cultureIn vitro screening in Al-amended solid media identifiedvariations in Al tolerance among the Pisolithus isolateswhereby Pisolithus that was isolated from abandoned andrestored mine sites was highly tolerant of Al (as Al3+) andPisolithus isolated from an adjacent native forest showedsignificantly lower tolerance to Al [9] Intersimple sequencerepeat (ISSR) fingerprinting also revealed strong intraspe-cific variation among these Pisolithus isolates (Egerton-Warburton unpublished data) These isolates were grown inbulk and then encapsulated in hydrogel beads [20] as thiscomprised an efficient method for the large-scale inoculationof containerized seedlings

22 Greenhouse Trial Two common forest trees used inrestoration Eucalyptus rudis Endl and Eucalyptus patensBenth were used in this study Seeds of each species werecollected frommature trees growing in abandonedmine siteson the lease of Western Collieries Ltd at Collie Half-sibling



4for 5min




4NO3) No further







3 Results



MineRestored

ForestControl

0

200

400

600

800

1000

1200

E patens E rudis

AA A

A AA

C B

Shoo

t mas

s (m

gmiddotpl

antminus1

)

(a)

E patens E rudis

A

A A A

B

B

C

D0

200

400

600

800

1000

1200

Shoo

t mas

s (m

gmiddotpl

antminus1

)

MineRestored

ForestControl

(b)

0

20

40

60

80

100

E patens E rudis

ECM

root

tips

()

A

B

C

B

C

A

B B

MineRestored

ForestControl

(c)









Biomass (pot)

N 0164 (009) 0004

269 684P 1075 (003) lt0001Ca 0858 (022) 0010Al minus0518 (007) 0008

Biomass (field)

N 0726 (008) 0004

271 586P 1990 (021) 0007Ca 0226 (002) 0005Mg 0275 (006) 0008Al minus0477 (004) 0002




4 Discussion






AAB

C

N co

nten

t (m

gmiddotpl

antminus1

)

0

2000

4000

6000

8000


C

AA

AB

0

100

200

300

400

500

600

P co

nten

t (m

gmiddotpl

antminus1

)


C

AAB

B

0

1000

2000

3000

K co

nten

t (m

gmiddotpl

antminus1

)


C

A A

B

0

1000

2000

3000Ca

cont

ent (

mgmiddot

plan

tminus1

)


Inoculum source

C

A

AB

B

0

500

1000

1500

2000

2500

Mg

cont

ent (

mgmiddot

plan

tminus1

)


(e)

C

A

BB


200

300

400

500

600

Al c

onte

nt (m

gmiddotpl

antminus1

)

(f)



E patens E rudis

MineForest

Host plant

Shoo

t mas

s (m

gmiddotpl

antminus1

)

lowast

lowast

0

100

200

300

400

500

600

(a)

K Ca Mg Al P

MineForest

Mineral nutrient

lowast

lowast

lowast

lowast

lowast

0

100

200

300

400

500

Nut

rient

cont

ent (

mgmiddot

plan

tminus1

)

(b)









5 Conclusions




Acknowledgments


References








































Journal of












Volume 2014

Advances in









Geophysics
















4for 5min




4NO3) No further







3 Results



MineRestored

ForestControl

0

200

400

600

800

1000

1200

E patens E rudis

AA A

A AA

C B

Shoo

t mas

s (m

gmiddotpl

antminus1

)

(a)

E patens E rudis

A

A A A

B

B

C

D0

200

400

600

800

1000

1200

Shoo

t mas

s (m

gmiddotpl

antminus1

)

MineRestored

ForestControl

(b)

0

20

40

60

80

100

E patens E rudis

ECM

root

tips

()

A

B

C

B

C

A

B B

MineRestored

ForestControl

(c)









Biomass (pot)

N 0164 (009) 0004

269 684P 1075 (003) lt0001Ca 0858 (022) 0010Al minus0518 (007) 0008

Biomass (field)

N 0726 (008) 0004

271 586P 1990 (021) 0007Ca 0226 (002) 0005Mg 0275 (006) 0008Al minus0477 (004) 0002




4 Discussion






AAB

C

N co

nten

t (m

gmiddotpl

antminus1

)

0

2000

4000

6000

8000


C

AA

AB

0

100

200

300

400

500

600

P co

nten

t (m

gmiddotpl

antminus1

)


C

AAB

B

0

1000

2000

3000

K co

nten

t (m

gmiddotpl

antminus1

)


C

A A

B

0

1000

2000

3000Ca

cont

ent (

mgmiddot

plan

tminus1

)


Inoculum source

C

A

AB

B

0

500

1000

1500

2000

2500

Mg

cont

ent (

mgmiddot

plan

tminus1

)


(e)

C

A

BB


200

300

400

500

600

Al c

onte

nt (m

gmiddotpl

antminus1

)

(f)



E patens E rudis

MineForest

Host plant

Shoo

t mas

s (m

gmiddotpl

antminus1

)

lowast

lowast

0

100

200

300

400

500

600

(a)

K Ca Mg Al P

MineForest

Mineral nutrient

lowast

lowast

lowast

lowast

lowast

0

100

200

300

400

500

Nut

rient

cont

ent (

mgmiddot

plan

tminus1

)

(b)









5 Conclusions




Acknowledgments


References








































Journal of












Volume 2014

Advances in









Geophysics















MineRestored

ForestControl

0

200

400

600

800

1000

1200

E patens E rudis

AA A

A AA

C B

Shoo

t mas

s (m

gmiddotpl

antminus1

)

(a)

E patens E rudis

A

A A A

B

B

C

D0

200

400

600

800

1000

1200

Shoo

t mas

s (m

gmiddotpl

antminus1

)

MineRestored

ForestControl

(b)

0

20

40

60

80

100

E patens E rudis

ECM

root

tips

()

A

B

C

B

C

A

B B

MineRestored

ForestControl

(c)









Biomass (pot)

N 0164 (009) 0004

269 684P 1075 (003) lt0001Ca 0858 (022) 0010Al minus0518 (007) 0008

Biomass (field)

N 0726 (008) 0004

271 586P 1990 (021) 0007Ca 0226 (002) 0005Mg 0275 (006) 0008Al minus0477 (004) 0002




4 Discussion






AAB

C

N co

nten

t (m

gmiddotpl

antminus1

)

0

2000

4000

6000

8000


C

AA

AB

0

100

200

300

400

500

600

P co

nten

t (m

gmiddotpl

antminus1

)


C

AAB

B

0

1000

2000

3000

K co

nten

t (m

gmiddotpl

antminus1

)


C

A A

B

0

1000

2000

3000Ca

cont

ent (

mgmiddot

plan

tminus1

)


Inoculum source

C

A

AB

B

0

500

1000

1500

2000

2500

Mg

cont

ent (

mgmiddot

plan

tminus1

)


(e)

C

A

BB


200

300

400

500

600

Al c

onte

nt (m

gmiddotpl

antminus1

)

(f)



E patens E rudis

MineForest

Host plant

Shoo

t mas

s (m

gmiddotpl

antminus1

)

lowast

lowast

0

100

200

300

400

500

600

(a)

K Ca Mg Al P

MineForest

Mineral nutrient

lowast

lowast

lowast

lowast

lowast

0

100

200

300

400

500

Nut

rient

cont

ent (

mgmiddot

plan

tminus1

)

(b)









5 Conclusions




Acknowledgments


References








































Journal of












Volume 2014

Advances in









Geophysics

















Biomass (pot)

N 0164 (009) 0004

269 684P 1075 (003) lt0001Ca 0858 (022) 0010Al minus0518 (007) 0008

Biomass (field)

N 0726 (008) 0004

271 586P 1990 (021) 0007Ca 0226 (002) 0005Mg 0275 (006) 0008Al minus0477 (004) 0002




4 Discussion






AAB

C

N co

nten

t (m

gmiddotpl

antminus1

)

0

2000

4000

6000

8000


C

AA

AB

0

100

200

300

400

500

600

P co

nten

t (m

gmiddotpl

antminus1

)


C

AAB

B

0

1000

2000

3000

K co

nten

t (m

gmiddotpl

antminus1

)


C

A A

B

0

1000

2000

3000Ca

cont

ent (

mgmiddot

plan

tminus1

)


Inoculum source

C

A

AB

B

0

500

1000

1500

2000

2500

Mg

cont

ent (

mgmiddot

plan

tminus1

)


(e)

C

A

BB


200

300

400

500

600

Al c

onte

nt (m

gmiddotpl

antminus1

)

(f)



E patens E rudis

MineForest

Host plant

Shoo

t mas

s (m

gmiddotpl

antminus1

)

lowast

lowast

0

100

200

300

400

500

600

(a)

K Ca Mg Al P

MineForest

Mineral nutrient

lowast

lowast

lowast

lowast

lowast

0

100

200

300

400

500

Nut

rient

cont

ent (

mgmiddot

plan

tminus1

)

(b)









5 Conclusions




Acknowledgments


References








































Journal of












Volume 2014

Advances in









Geophysics















AAB

C

N co

nten

t (m

gmiddotpl

antminus1

)

0

2000

4000

6000

8000


C

AA

AB

0

100

200

300

400

500

600

P co

nten

t (m

gmiddotpl

antminus1

)


C

AAB

B

0

1000

2000

3000

K co

nten

t (m

gmiddotpl

antminus1

)


C

A A

B

0

1000

2000

3000Ca

cont

ent (

mgmiddot

plan

tminus1

)


Inoculum source

C

A

AB

B

0

500

1000

1500

2000

2500

Mg

cont

ent (

mgmiddot

plan

tminus1

)


(e)

C

A

BB


200

300

400

500

600

Al c

onte

nt (m

gmiddotpl

antminus1

)

(f)



E patens E rudis

MineForest

Host plant

Shoo

t mas

s (m

gmiddotpl

antminus1

)

lowast

lowast

0

100

200

300

400

500

600

(a)

K Ca Mg Al P

MineForest

Mineral nutrient

lowast

lowast

lowast

lowast

lowast

0

100

200

300

400

500

Nut

rient

cont

ent (

mgmiddot

plan

tminus1

)

(b)









5 Conclusions




Acknowledgments


References








































Journal of












Volume 2014

Advances in









Geophysics















E patens E rudis

MineForest

Host plant

Shoo

t mas

s (m

gmiddotpl

antminus1

)

lowast

lowast

0

100

200

300

400

500

600

(a)

K Ca Mg Al P

MineForest

Mineral nutrient

lowast

lowast

lowast

lowast

lowast

0

100

200

300

400

500

Nut

rient

cont

ent (

mgmiddot

plan

tminus1

)

(b)









5 Conclusions




Acknowledgments


References








































Journal of












Volume 2014

Advances in









Geophysics
















5 Conclusions




Acknowledgments


References








































Journal of












Volume 2014

Advances in









Geophysics






























Journal of












Volume 2014

Advances in









Geophysics


















Journal of












Volume 2014

Advances in









Geophysics














Documents

Pisolithus Eucalyptus - Hindawi Publishing Corporationdownloads.hindawi.com/journals/aess/2015/803821.pdfEucalyptus seedlings were inoculated with cultures of Pisolithus previously