Increased plant tolerance against chrysanthemum yellows

phytoplasma (‘Candidatus Phytoplasma asteris’) following

double inoculation with Glomus mosseae BEG12 and

Pseudomonas putida S1Pf1Rif1; 21; 2

R. D’Amelioab, G. Bertac, E. Gamaleroc, N. Massac, L. Avidanoc, S. Cantamessac,

G. D’Agostinoc, D. Boscob and C. Marzachıa*aCNR, Istituto di Virologia Vegetale, Strada delle Cacce 73, Torino 10135; bUniversita di Torino, Dipartimento di Valorizzazione e

Protezione delle Risorse Agroforestali, Via Leonardo Da Vinci 44, Grugliasco (Torino) 10095; and cUniversita del Piemonte Orientale

‘Amedeo Avogadro’, Dipartimento di Scienze dell’Ambiente e della Vita, Viale Teresa Michel 11, Alessandria 15121, Italy

The aim of this work was to assess the effects of a combined inoculum of a rhizobacterium and an arbuscular mycorrhizal

(AM) fungus on plant responses to phytoplasma infection, and on phytoplasma multiplication and viability in Chrysanthe-

mum carinatum plants infected by chrysanthemum yellows phytoplasma (CY). Combined inoculation with Glomus mos-

seae BEG12 and Pseudomonas putida S1Pf1Rif resulted in some resistance to phytoplasma infection (about 30%), delayed

symptom expression in nonresistant plants, improved growth of the aerial part of the infected plants (+68Æ1%), and altered

root morphology (root tip number: +49Æ9%; branching degree: +82Æ8%). Combined inoculation with the two beneficial

microorganisms did not alter CY multiplication and viability. In inoculated and infected plants, phytoplasma morphology

was typical of senescent cells. A more active and efficient root system in double-inoculated plants probably mediated the

effects of the two rhizospheric microorganisms in the infected plants. The practical application of rhizospheric microorgan-

isms for mitigating phytoplasma damage, following evaluation under field conditions, represents an additional tool for the

integrated management of phytoplasmosis.

Keywords: arbuscular mycorrhizal fungus, Chrysanthemum carinatum, chrysanthemum yellows phytoplasma

infection, rhizobacteria

Introduction

Phytoplasmas are wall-less, nonhelical prokaryotes thatcolonize plant phloem and insects. They are associatedwith more than 600 diseases, some of great economicimportance, in several hundred plant species (Seemulleret al., 1998),with symptoms such aswitches’ broom, pro-liferation, flower malformation (virescence, phyllody),stunting and general decline.Phytoplasmas are transmitted by phloem-sucking

insects, mostly in the families Cicadellidae, Cixiidae andPsyllidae, in a persistent, propagativemanner (Weintraub& Beanland, 2006). Control of phytoplasma diseases ismainly achieved by insecticide treatments against the vec-tors, with deleterious impacts on the environment. Infruit trees, where economic lossesmay be severe, breedingfor resistant varieties and hot water treatment of propa-gation material are currently implemented in controlstrategies. The exploitation of the natural recovery of

some fruit tree species, such as grapevine and apple, isalso suggested by several research groups as a possibletool for mitigating phytoplasma impacts (Romanazziet al., 2009), although the control of insect vector popula-tions is required to prevent re-infections (Morone et al.,2007). In other cases, such as in the Bois noir infection ofgrapevine, alternative strategies for controlling the phy-toplasma are even more important. In this case, since theknown vector of the phytoplasma, Hyalesthes obsoletus(Maixner, 1994), does not breed on grapes, planting ofhealthy propagation material and management of weedsin the vineyard are the only availablemeasures of control-ling the disease.The potential role of arbuscular mycorrhizal (AM)

fungi and rhizosphere bacteria as defence elicitors hasbeendescribed (vanLoon et al., 1998;Akhtar&Siddiqui,2008). Arbuscular mycorrhizal fungi form mutualisticassociations with most terrestrial plants, includingagricultural and horticultural crop species. They obtaincarbohydrates from the host plant, and, in return, theyassist the plant in the acquisition of mineral nutrients(mainly phosphorus) and water (Smith & Read, 2008),and they also modify root architecture and topology

P P A 2 4 7 9 B Dispatch: 19.5.11 Journal: PPA CE: Kannan

Journal Name Manuscript No. Author Received: No. of pages: 9 PE: Prasanna

*E-mail address: c.marzachi@ivv.cnr.it

ª 2011 The Authors

Plant Pathology ª 2011 BSPP 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

Plant Pathology (2011) Doi: 10.1111/j.1365-3059.2011.02479.x

(Hodge et al., 2009). This symbiosis not only influencesplant nutrition, but it also improves the plant’s ability toovercome biotic (Lingua et al., 2002; Berta et al., 2005)and abiotic (Volante et al., 2005; Gamalero et al., 2010a)stresses. Moreover, the synthesis of volatile compounds(Guerrieri et al., 2004) and the secondary metabolicpathways of plants (Copetta et al., 2006) are affected inAMplants.Bacteria living on the roots may promote plant

growth via a number of mechanisms, including hormonesynthesis, phosphate solubilization, nitrogen fixation(Gamalero & Glick, 2011) and root architecture modifi-cations (Gamalero et al., 2002, 2004, 2008). However,the beneficial effect of these bacteria also relies on theimprovement of plant health through the suppression ofsoilborne diseases. In this context, the production of anti-biotics, lytic enzymes and siderophores, associated withan increase in plant tolerance to environmental stresses,play a key role. Besides affecting plant growth and health,bacteria living in the rhizosphere can interact with AMfungi. Stimulation of the AM symbiosis developmentby rhizospheric bacteria has been reported (Ravnskov &Jakobsen, 1999;Gamalero et al., 2004, 2008; Pivato et al.,2009). Although knowledge of the mechanism involvedin this positive plant ⁄bacteria ⁄AM fungi multitrophicinteraction is still incomplete, synthesis of the enzyme1-aminocyclopropane-1-carboxylate (ACC) deaminase,which lowers the levels of ethylene in plants, seems to beinvolved (Gamalero et al., 2008). On the other hand,bacteria and AM fungi inoculated together can inducesynergistic effects onplants (Gamalero et al., 2008).Increased tolerance against phytoplasmas has been

demonstrated in plants inoculated with AM fungi (Lin-gua et al., 2002; Garcıa-Chapa et al., 2004; Kaminskaet al., 2010) or with bacteria colonizing plant roots(Gamalero et al., 2010b). The possible synergistic effecton phytoplasma infection of concurrent inoculation withAM fungi and beneficial rhizospheric bacteria has neverbeen addressed. Therefore, the aim of this work was toassess the effects of a mixed inoculum, composed of arhizobacterium and an AM fungus on plant responses tophytoplasma infection and on phytoplasma multiplica-tion and viability in the model plant Chrysanthemumcarinatum infected by ‘Candidatus Phytoplasma asteris’chrysanthemumyellows (CY).

Materials and methods

Microorganisms

The strain Pseudomonas putida S1Pf1 was isolated fromthe soil next to a symptomless grapevine (Vitis viniferacv. Barbera) among plants with clear symptoms ofphytoplasma infection (D’Amelio et al., 2007). A spon-taneous rifampicin-resistant mutant (S1Pf1Rif) wasobtained, grown in Luria-Bertani (LB) broth supple-mented with rifampicin (100 lg mL)1) and stored at)80�C in 50% glycerol. This bacterial strain, unable toproduce ACC deaminase, was able to produce sidero-

phores and auxin, to solubilize phosphate and to relievesymptoms in chrysanthemums infected byCY (Gamaleroet al., 2010b).The AM fungus Glomus mosseae BEG12 was previ-

ously shown to promote plant growth (Gamalero et al.,2002, 2004; Kaminska et al., 2010) and to suppresssoilborne diseases caused by Phytophthora parasitica(Trotta et al., 1996), Rhizoctonia solani (Berta et al.,2005;Gamalero et al., 2010b) and phytoplasmas (Linguaet al., 2002). A mycorrhizal inoculum consisting ofinfected leek roots, sporocarp spores and hyphae ofG. mosseae BEG12 on quartz sand was obtained fromAgrauxine-Biorize.

Plant growth conditions and treatments

Chrysanthemum carinatum seeds were surface-sterilizedby gently shaking in a 50-g L)1 sodiumhypochlorite solu-tion for 3 min. The seeds were rinsed six times for 5 minand four times for 20 min in sterile deionized water,placed in Petri dishes on moist sterile filter paper andincubated in the dark at 24�C for 3 days. The efficiency ofthe sterilization procedure was assessed by placing 20C. carinatum seeds on nutrient agar (NA, Fluka) for3 days. Chrysanthemum carinatum plants were grownfrom sterilized seeds in 8 · 8-cm pots containing a mix-ture of 0Æ6–1Æ2 mm coarse grade quartz sand (Punto Elle)sterilized at 200�C for 2 h. The plants were grown in agreenhouse (25�C, 16-h photoperiod) andwatered to sat-uration with a modified Long Ashton nutrient solutioncontaining 32 lM phosphate three times perweek (Trottaet al., 1996).Inoculationwith a commercial inoculumofG.mosseae

BEG12 was performed by potting C. carinatum seeds inthe sterile mix of quartz and sand together with100 g L)1 of commercial inoculum.Pseudomonas putida S1Pf1Rif inoculants were pro-

duced on King’s B (KB) agar plates with 100 lg mL)1

rifampicin added at 28�C for 48 h. Bacteria were scrapedfrom themedium, suspended in 0Æ1 MMgSO4, pelleted bycentrifugation (4500 g, 20 min), washed twice and sus-pended in the same buffer. The bacterial density of thesuspension was determined using a calibration curveassessed by measuring optical density at k = 600 nm,and then adjusted to 1010 colony forming units mL)1

(CFU mL)1). The bacterial inoculumwas applied 15 daysafter sowing bywatering the germinated plants with 5 mLof a suspension (1010 CFU mL)1) of P. putida S1Pf1Rifin order to reach the final density of 108CFU g)1of sand.The control plants were sown in the sterile mix of

quartz and sand devoid of G. mosseae BEG12 inoculumand watered with 5 mL sterile 0Æ1 M MgSO4 instead ofthe bacterial inoculum.There were four treatments and 10–20 replicates per

treatment: control plants (C), CY-infected plants (CY),P. putida S1Pf1Rif- and G. mosseae BEG12-inoculatedplants (Pp + Gm)andP.putida S1Pf1Rif- andG.mosseaeBEG12-inoculated and CY-infected plants (Pp + Gm +CY). The experiment was repeated three times (a total of

2 R. D’Amelio et al.

Plant Pathology (2011)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

42 and 49 non-inoculated and inoculated plants, respec-tively) and the results presented here are the mean valuesof the data obtained from these independent experi-ments.

Phytoplasma and vector insect

‘Candidatus Phytoplasma asteris’, strain CY, originallyisolated from Argyranthemum frutescens plants inLiguria (Italy) (Conti et al., 1988), was maintained inC. carinatum by vector transmission.Healthy colonies of Macrosteles quadripunctulatus

were reared on potted oat plants inside plexiglass andnylon cages in growth chambers at 25�C, with a 16-hphotoperiod, and checked by polymerase chain reaction(PCR) assays to verify the absence of phytoplasma.For the transmission experiments, third to fifth instar

nymphswere fed for 1 weekonCY-infected plants, trans-ferred to healthy oat plants for 2 weeks to completelatency, and then singly transferred to 10 ⁄15 C. carina-tum for each elicitor and control treatment for aninoculation access period (IAP) of 3 days inside glasscylinders. Inoculation with single vectors was performedin order to reduce the concentration of the initial inocu-lum in the plant. Test plants were exposed to vectors2 months after sowing. The plants were then treatedwith insecticides and maintained in the greenhouse for1 month. Chrysanthemum carinatum exposed to vectorswere used as transmission controls and others inoculatedwith P. putida S1Pf1Rif and G. mosseae BEG12 but notexposed to vectorswere used as treatment controls.

Evaluation of plant development and rootarchitecture

Ten plants per treatment were collected 3 months aftersowing (at 30 dpi) and the following parameters weredetermined: plant, root and shoot fresh weights, root ⁄shoot fresh weight ratio, total root length, total surfacearea, number of tips, and degree of root branching (repre-sented by root tip number divided by total root length),by using a dedicated Desk Scan II scanner equippedwith a special lighting system for rootmeasurements. Thedigitalized root images were analysed by the WINRHIZO

software (Regent Instruments).

Bacterial density and mycorrhizal colonization

Bacterial density was assessed 2Æ5 months after sowing(at 30 dpi). Whole root systems were aseptically cut fromfive plants, gently washedwith sterile water and vortexedfor 15 min in MgSO4 buffer (0Æ1 M). The suspensionsobtained were serially diluted and plated on solid KBsupplemented with rifampicin (100 lg mL)1). Afterincubation for 48 h at 25�C, the numbers of CFU g)1

root freshweightwere determined.The degree of mycorrhizal colonization within the

root systems was evaluated according to Trouvelot et al.(1986). At least 30 randomly chosen, 1-cm-long root

fragments were cleared for 20 min at 60�C in 100 g L)1

KOH and stained with 10 g L)1 methyl blue in lacticacid. Root fragments were mounted on glass slides andexamined microscopically, with magnifications up to·630. The mycorrhizal frequency (F%) was calculated asthe ratio between root fragments colonized byAM fungalmycelium and the total number of root fragments analy-sed. The mycorrhizal colonization intensity (M%) wasevaluated as the amount of root cortex colonized by thefungus relative to the whole root system. Arbuscule andvesicle richness in the whole root system were measuredby arbuscule (A%) and vesicle (V%) abundance.

Symptom evaluation

The presence and severity of CY symptoms on the plantswere evaluated every 3 days starting from 9 up until30 days after the beginning of inoculation (dpi), andplants were classified into five classes of severity: 0 = nosymptoms, 1 = yellowing of the apex, 2 = yellowing anddistortion of the apex, 3 = stunted apex growth,4 = severe yellowing and dwarfing of the whole plant,5 = plant death.

Phytoplasma concentration: DNA extraction andquantitative real-time PCR

Samples of 200 mg were collected from the apical leavesof five plants per treatment at 17 and 24 dpi, and totalDNA was extracted from 100 mg using the PureLinkPlant Total DNA Purification kit (Invitrogen), accordingto the manufacturer’s protocol. The same plants weresampled on both dates and quantification was repeatedtwice (experimental repeats 1 and 2). The concentrationof DNA in the extracts was measured using a NanoDrop1000 Spectrophotometer (Thermo Fisher Scientific).Diluted samples (1 ng lL)1 in sterile double-distilledwater) were analysed in triplicate by quantitative real-time PCR (Q-PCR) assays. The CY DNA in each samplewas measured as the number of CY genome units (GU)ng)1 of plant DNA, as described by Marzachi & Bosco(2005). The DNA from healthy plants and the PCR mix-ture devoid of a template were used as negative controls.Threshold cycles and standard curves were automaticallycalculated by the BIORAD ICYCLER software, version3Æ06070. The CY and host DNA from the same samplewere quantified in the same plate.

Phytoplasma viability: total RNA extraction andquantitative reverse transcription real-time PCR

The remaining 100 mg leaf tissue collected from the fiveplants per treatment of experimental repeats 2 and 3 forDNA quantification were ground to powder under liquidnitrogen. Total RNA, extracted with Trizol Reagent(Invitrogen) and treated with RQI RNase-free DNase(Promega), was loaded as the template in quantitativereverse transcription real-time PCR (qRT-PCR) assaysas described by D’Amelio et al. (2010). In the control

Effects of AM fungus and a pseudomonad on phytoplasmosis 3

Plant Pathology (2011)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

reactions, RNA samples were not supplemented withreverse transcriptase to rule out DNA contamination.The RNA from healthy plants and the PCR mixturedevoid of the template were used as negative controls. Allsampleswere run in triplicate.The phytoplasma 16S rDNA transcript copy number

per nanogram of total RNA of each sample was deter-mined.Thephytoplasma16S rDNA transcript copy num-ber per 100 mg fresh leaf tissue was then derived. Theviability of CY (expressed as 16S rDNA transcripts perphytoplasma cell) was estimated by dividing the tran-script copy number by the number of CY cells in 100 mgof the same sample (measured as detailed above).

Microscopic observations

Pieces of the basal, median and apical zones of matureleaves sampled along the entire stem of plants infectedwith CY or infected with CY and inoculated withP. putida S1Pf1R1 andG.mosseae BEG12were fixed for3 h at room temperature in 3% glutaraldehyde in 0Æ15 M

phosphate buffer (pH7Æ2) and post-fixed for 2 h at 4�C in10 g L)1 osmium tetroxide, in the same buffer. The sam-ples were stained overnight at 4�C in 10 g L)1 aqueousuranyl acetate, dehydrated in an ethanol series andembedded in Epon-Araldite. Pieces of leaves from theuntreated plants were processed in the same way as thecontrols. Ultra-thin sections cutwith a diamondknife in aLeika Ultracut UCT ultramicrotome (Leica Mikrosys-teme Gmbh) were stained with lead citrate and examinedunder a Philips CM10 transmission electronmicroscope.About 15 sectionswere observed for each treatment.

Statistical analysis

The data on plant growth and root architecture obtainedfrom 10 plants per treatment were expressed asmean val-ues. The data onmycorrhizal colonizationwere subjectedto angular transformation before statistical analysis.Nontransformed and transformed values were submittedto analysis of variance and to Fisher’s least significant test(P £ 0Æ05) using the STATVIEW 5Æ0 statistics package (SASInstitute Inc.).In order to analyse symptom development in control

and elicited plants, the median number of days requiredby each plant to reach each symptom severity class wascalculated and then compared using the non-parametricMann–Whitney U Test using SIGMAPLOT 11 (Systat Soft-ware Inc.).Raw data (obtained as a log) of the phytoplasma titre

were used and the concentration of CY phytoplasma ineachplantwasexpressed as the difference between the log-arithmic concentrations of CY and C. carinatum DNAs.Inorder to compare the phytoplasma titremeasured at dif-ferent dpi in elicited and control plants, a two-way ANOVA

for date and treatment was performed (SIGMAPLOT 11Æ0;Systat Software Inc.).Raw data of phytoplasma viability (CY phytoplasma

transcripts ⁄CYphytoplasma cells)were transformed into

logarithms since the standard deviation appeared as afunction of the mean. Two-way ANOVA (treatment anddate) was performed to compare the CY viability mea-sured on the two sampling dates (17 and 24 dpi) inCY-infected plants inoculated or not with P. putidaS1Pf1Rif and G. mosseae BEG12 (JANDEL SIGMAPLOT

11Æ0; Systat Software Inc.).

Results

Bacterial density and mycorrhizal colonization

At the end of the experiments, the density of P. putidaS1Pf1Rif was significantly higher in CY-infected plantsthan in the uninfected ones (5Æ05 ± 0Æ74 and 4Æ01 ± 0Æ13log CFU g)1 root fresh weight, respectively; P = 0Æ002),and the root systems were densely colonized by G. mos-seae BEG12. Infection with CY did not significantlyaffect mycorrhizal frequency, colonization intensity orarbuscule abundance (not shown). However, vesicleabundance was significantly reduced (P = 0Æ006) inCY-infected plants (9Æ7 ± 2Æ9) compared to the controls(21Æ7 ± 1Æ5). No mycorrhizal colonization was observedin control or phytoplasma-infected, non-inoculatedplants.

Effect of the microorganisms on plant growth, rootarchitecture and symptom development

Plant growth was reduced by CY ()37Æ3%, Fig. 1a,P = 0Æ0023). Inoculationwith the bacterial strain and theAM fungus increased the biomass of infected plants com-pared to the controls (+60Æ5%, Fig. 1a, P = 0Æ0305) andrescued the plant growth depression induced by CY(Fig. 1a, P = 0Æ0024). In particular, root and shoot freshweights in CY-infected plants were reduced by 39Æ4 and34Æ8%, respectively, compared to the controls (Fig. 1b,c,P = 0Æ0070 and P = 0Æ0146, respectively). The shootfreshweight ofCY-infected plants inoculatedwithP. put-ida S1Pf1Rif and the mycorrhizal fungus was signifi-cantly higher (P < 0Æ0001) than that of CY-infectedplants (+68Æ1%) (Fig. 1c). By contrast, inoculation withthe two beneficial microorganisms did not rescue the CY-induced reduction of root freshweight (Fig. 1b). The ratiobetween root and shoot fresh weights was reduced inplants inoculated with S1Pf1Rif andG. mosseae BEG12,infected ()45Æ1%, P = 0Æ0113) or not with CY ()54Æ4%,P = 0Æ0001) compared to the controls (Fig. 1d).All of the root architecture parameters were reduced

in plants infected by CY. Inoculation with the strainS1Pf1Rif and the AM fungus in plants infected by thephytoplasma increased the root tip number (+49Æ9%,P = 0Æ009) and the branching degree (+82Æ8%, P <0Æ001) compared to the non-inoculated and infectedcontrols (Table 1).Symptom development was evaluated every 3 days for

a period of 30 days, starting from 9 dpi. Pseudomonasputida S1Pf1Rif and G. mosseae BEG12-inoculatedand non-inoculated control plants did not develop any

4 R. D’Amelio et al.

Plant Pathology (2011)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

symptoms (data not shown). Following exposure to CY-infective leafhoppers, a higher number of elicited plantsthan non-inoculated ones did not develop symptoms(13 ⁄49 vs. 3 ⁄42; v2 = 4Æ605;P = 0Æ032).The results from the three experimental repeats were

pooled for analysis since a one-way ANOVA on ranks didnot reveal any differences between the repeats for bothcontrol and inoculated plants. The median numbers ofdays required by elicited plants to reach the differentsymptom severity classes were higher than for the controlplants, although these differences were not significant(not shown).

Chrysanthemum yellows phytoplasma titre, viabilityand phytoplasma morphology

The phytoplasma titre and viability in CY-infectedplants, inoculated or not with the fluorescent pseudomo-nad and the AM fungus, were measured on apical leavesat 17 and 24 dpi. Two-way ANOVA for treatment and dateshowed that the CY titre increased significantly over time

(P = 0Æ002), whereas no differences were found betweenelicited and control (P = 0Æ990) plants (Table 2). Theinteraction between the two factors (inoculation with theelicitors and sampling date) did not affect the CY titre.The numbers of 16S rDNA transcripts per CY cell (as anestimator of CY viability) in inoculated and CY-infectedplants, as well as in the controls, are reported in Table 2.Two-way ANOVA for treatment and date showed a highlysignificant reduction in phytoplasma viability between 17and 24 dpi (P < 0Æ001), whereas no differences werefound between inoculated and control plants(P = 0Æ186). TheCYviabilitywas unaffected by the inter-action between the two factors (inoculationwith the elici-tors and sampling date).Phytoplasmas were never detected in the plants inocu-

lated with both beneficial microorganisms or the controlplants that were not exposed to infective insects (data notshown).Microscopic observations detected differences in

phytoplasma morphology according to plant treatment.No phytoplasma cells were detected in control plants or

(a) (b)

(c) (d)

Figure 1 Effect of microorganisms on

growth and root architecture of

Chrysanthemum carinatum plants. Plant

(a), root (b), shoot (c) fresh weight and

root ⁄ shoot fresh weight ratio (d) of

control plants, plants infected with

chrysanthemum yellows (CY) and plants

inoculated with Pseudomonas putida

S1Pf1Rif and Glomus mosseae BEG12,

infected or not infected with CY. Means

with the same letter are not significantly

different (P £ 0Æ05) according to Fisher’s

least significant difference test.

Table 1 Total root length (TL), total root surface

area, number of tips (T), and root branching

degree (T ⁄ TL) inChrysanthemum carinatum

plants inoculated or not with Pseudomonas

putida S1Pf1Rif andGlomus mosseae BEG12

(C, Pp + Gm) and infected or not with CY (CY,

Pp + Gm + CY)

TL TSA T T ⁄ TL

C 4414Æ7 ± 348Æ9 a 560Æ0 ± 35Æ0 a 5974Æ8 ± 397Æ8 a 1Æ4 ± 0Æ1 b

CY 2472Æ9 ± 96Æ9 c 335Æ1 ± 16Æ0 b 2503Æ0 ± 186Æ3 c 1Æ0 ± 0Æ1 c

Pp + Gm 3371Æ3 ± 381Æ1 b 526Æ5 ± 50Æ0 a 3984Æ3 ± 293Æ3 b 1Æ2 ± 0Æ1 bc

Pp + Gm + CY 2041Æ7 ± 131Æ9 c 264Æ9 ± 19Æ1 b 3751Æ6 ± 296Æ1 b 1Æ8 ± 0Æ2 a

Values are means of 10 plants ± standard errors. Within columns, means with the same letter

are not significantly different (P £ 0Æ05) according to Fisher’s least significant difference test.

The analyses were performed at the end of the experiments (30 dpi).

Effects of AM fungus and a pseudomonad on phytoplasmosis 5

Plant Pathology (2011)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

in those inoculated with the AM fungus and P. putidaS1Pf1Rif. The deposition of callose in the pore of the sieveelements and the synthesis of PR protein in the host plantwas found in CY-infected plants (Fig. 2a,b). In general, inthese plants, the phytoplasmas were densely pressed intothe phloem lumen in all of the observed samples. In CY-infected plants inoculated with G. mosseae BEG12 andthe bacterial strain S1Pf1Rif (Fig. 2c), the phytoplasmamorphology (empty cells and cells with the cytoplasmconfined to the periphery) was typical of senescent cells,clearly indicating the occurrence of a strong degenerativeprocess.

Discussion

This work shows the effects of a combination of two rhi-zospheric microorganisms on phytoplasma infection of amodel plant. Plant growth, root architecture, multiplica-

tion of the phytoplasma and its metabolic activity weremeasured in ‘Candidatus Phytoplasma asteris’ (chrysan-themumyellows strain, CY)-infectedC. carinatum plantsinoculated with P. putida S1Pf1rif (Gamalero et al.,2010b) and the AM fungusG.mosseaeBEG12, and com-pared to CY-infected but non-inoculated plants. Previousworks demonstrated the activity of P. putida S1Pf1Rif onC. carinatum infected by CY (Gamalero et al., 2010b)and of G. mosseae BEG12 on tomato plants infected bythe stolbur phytoplasma (Lingua et al., 2002) andCatha-ranthus roseus infected by a ‘Ca. Phytoplasma asteris’strain (Kaminska et al., 2010). Nevertheless, this is thefirst report dealing with the activity of a combination oftwo plant-beneficial microorganisms on a phytoplasma-infected plant.Inoculation with the two rhizospheric microorganisms

resulted in a lower number of infected plants followingexposure to infective leafhopper vectors. Protection of

Table 2 Mean chrysanthemum yellows (CY) phytoplasma titre (CY cells per nanogram plant DNA ± standard error) and viability (mean copy number of CY

16S rDNA transcripts per CY cell ± standard error) in CY-infected Chrysanthemum carinatum (CY) inoculated or not with Pseudomonas putida S1Pf1Rif and

Glomus mosseae BEG12 (Pp + Gm + CY). Plants were sampled at 17 and 24 days post-inoculation (dpi)

Sampling

date (dpi)

CY cells ⁄ ng plant DNA (mean ± SE) CY 16S rDNA transcripts ⁄CY cell

CY Pp + Gm + CY CY Pp + Gm + CY

17 146 500 ± 30 284 a 149 973 ± 39 109 a 4455 ± 946 A 1207 ± 349 B

24 324 000 ± 60 482 b 521 714 ± 152 437 b 1207 ± 349 B 1726 ± 430 B

Within rows and columns of each experiment, means followed by the same letter do not differ significantly.

(a)

(c)

(b)

Figure 2 Phytoplasma morphology.

Micrographs of Chrysanthemum carinatum

leaves (a, b) infected with chrysanthemum

yellows (CY) phytoplasma and (c) infected

with CY and inoculated with Pseudomonas

putida S1Pf1Rif and Glomus mosseae

BEG12. In the phloem of CY-infected control

plants, the CY cells are well preserved

(a and b), bound by a well-defined unit

membrane and contain a matrix of varying

electron densities; copious formation of

electron-transparent callose pinching off the

pores is evident in the sieve plates (b, black

arrows). In the phloem of plants infected with

CY and inoculated with P. putida S1Pf1Rif

and G. mosseae BEG12, few and

degenerated forms of CY (c, black arrows)

are evident. Bar = 1 lm.

6 R. D’Amelio et al.

Plant Pathology (2011)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

plants from CY infection was not observed in C. carina-tum inoculated with P. putida S1Pf1Rif alone (Gamaleroet al., 2010b) or elicited with benzothiadiazole (D’Ame-lio et al., 2010). In preliminary experiments, inoculationofC. carinatumwithG.mosseaeBEG12 alone resulted ina reduced number of CY-infected plants following expo-sure to infective hoppers (authors’ unpublished results).Therefore, it can be speculated that resistance to CYinfection can be attributed to the activity of themycorrhi-zal fungus. In a different phytoplasma–plant association,the inoculation of periwinkles with G. mosseae BEG12alone did not affect the number of ‘Ca. Phytoplasmaasteris’-infected plants (Kaminska et al., 2010); however,in that experimental setup, the phytoplasma was trans-mitted by grafting.The CY infection induced a severe reduction in plant

growth, both at the root and the shoot level. Inoculationwith the AM fungus and the bacterial strain induced asignificant increase in the fresh weight of the aerial partof the plant, and this effect was still present upon CYinfection. According to the results presented by Gamale-ro et al. (2010b) the bacterial strain did not improveplant growth. Therefore, the increase of the shoot bio-mass in plants inoculated with G. mosseae BEG12 andP. putida S1Pf1Rif may be ascribed to the fungal partneror to the effects resulting from the interactions betweenthe two beneficial microorganisms (Gamalero et al.,2004, 2008). Moreover, the reduction in the freshweight root ⁄ shoot ratio in inoculated plants, irrespectiveof phytoplasma infection, suggests that the net benefit ofthe mycorrhizal association largely exceeded its net cost,as already reported by Fitter (1991). Similarly, G. intra-radices mycorrhizal infection of pear trees was associ-ated with a significant increase in plant shoot length inpear decline-infected and healthy controls (Garcıa-Chapa et al., 2004). The increase in the development ofthe aerial part of the plant was supported by a largerroot system in the double-inoculated plants. Indeed,double-inoculated, CY-infected plants showed not onlya larger root system but also a higher degree of rootbranching (Table 1, T ⁄TL), thus allowing the explora-tion of a higher volume of soil than other treatments.This difference would account for the increased growthof the double-inoculated CY-infected plants, as mea-sured by plant weight. The CY infection caused a signifi-cant reduction in the number of root tips (Table 1, T).This reduction was also present, although to a lesserextent, in the elicited but healthy controls, and the pres-ence of the two elicitors resulted in an increase in thenumber of root tips in the infected plants. The CY infec-tion also induced a severe reduction in the degree of rootbranching (Table 1, T ⁄TL) in control plants, but rootsof double-inoculated CY-infected plants were morebranched than those in any of the other treatments.Treatment of C. carinatum plants with P. putidaS1Pf1rif alone did not rescue the reduced root branchingassociated with CY infection (Gamalero et al., 2010b),thus suggesting a relevant role of the AM fungus in thepromotion of lateral root initiation. In fact, increased

branching seems to be a more general effect induced byAM fungi (Berta et al., 1995, 2002, 2005; Hodge et al.,2009), leading to better nutrient absorption, mainlyphosphate, from the soil.Following inoculation with the fluorescent pseudomo-

nad and the AM fungus, CY-infected plants showed aconstant, although not significant, delay in symptomdevelopment compared to control plants. Although it isevident that symptom severity does not provide a directmeasure of the agronomic losses associated with phyto-plasma infection, it still represents a measure of theimpact of the pathogen on plant health.Phytoplasma multiplication in apical leaves was not

affected by P. putida S1Pf1rif and G. mosseae BEG12.Consistent with this, single inoculations with P. putidaS1Pf1rif (Gamalero et al., 2010b) or G. mosseae alone(author’s unpublished results) also had no effect on themultiplication of CY. Phytoplasma viability (as deter-mined by the number of 16S rDNA transcripts per CYcell) in the same plant tissues decreased with the progres-sion of the disease and no differenceswere found betweenthe inoculated and non-inoculated plants. On the otherhand, phytoplasma cells in mature leaves were degener-ated in double-inoculated plants when observed byTEM;this observation correlates with the delay in symptomdevelopment in inoculated plants. Recently, hormonaland transcriptional profile analyses indicated the roleof G. mosseae in the jasmonate biosynthetic pathway(Lopez-Raez et al., 2010) in root tissues. It can be specu-lated that the activation of metabolic pathways involvedin plant resistance to biotic stresses is involved in thesenescence of phytoplasma cells in fully differentiatedandmature leaves, and that has aminor effect on dividingand elongating cells of apical leaves, possibly because oftheir distance from the roots (Lopez-Raez et al., 2010).This could explain why viable CY cells were found indeveloping apical leaves of double-inoculated plantsin the present study. Therefore, for the two combinedmicroorganisms, an indirect effect on phytoplasma infec-tion and on the relief of phytoplasma damage, perhapsnot just related to the improved nutritional status of inoc-ulatedC. carinatum, is envisaged.At the end of the experiments, both the fluorescent

pseudomonad and the AM fungus were present andabundant on and inside the plant roots, respectively.Actually, the density and persistence of P. putidaS1Pf1Rif was higher in CY-infected plants compared toinoculated healthy controls. On the other hand, only thefrequency of vesicles (Table 2, V%), among all of themycorrhizal colonization parameters, was lowered inCY-infected plants. Vesicles are lipids and cytoplasm-containing hyphal swellings useful when the plant ismaturing or is exposed to unfavourable conditions (Smith&Read, 2008), as is the case in phytoplasma infections.In conclusion, the application of a combination of an

AM fungus and a rhizospheric bacterium provided twomajor results: some level of resistance (about 30%) tophytoplasma infection, and increased tolerance to thedisease as measured by delayed symptom expression and

Effects of AM fungus and a pseudomonad on phytoplasmosis 7

Plant Pathology (2011)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

improved growth of infected plants. The toleranceinduced by the combination of the two microorganismswas probably not just mediated by the improved growthof the plant and might be associated with alterations inthe hormonal balance of the inoculated plant (Lopez-Raez et al., 2010).Based on these results from an experimental patho-

system model, the practical application of rhizosphericmicroorganisms for mitigating phytoplasma damageshould be evaluated under field conditions, especially inthe case of perennial crops. This method might representan additional tool for the integrated management ofphytoplasmosis, and it might eventually allow a reduc-tion in insecticide applications for more environmentallyfriendly control of these diseases.

Acknowledgements

This workwas supported by the Piedmont Region withinthe project ‘Valutazione dell’azione di microrganismirizosferici ed elicitori di resistenza sull’infezione da fito-plasmi in un sistemamodello (CIPE 2006)’.

References

AkhtarMS, Siddiqui ZA, 2008. Arbuscular mycorrhizae fungi as

potential bioprotectans against plant pathogens. In: Siddiqui

ZAA, SayeedM, Kazuyoshi F, eds.Mycorrhizae: Sustainable

Agriculture and Forestry. Dordrecht, the Netherlands: Springer

Publisher, 362.

Berta G, Trotta A, Fusconi A et al., 1995. Arbuscular mycorrhizal

induced changes to plant-growth and root-system morphology in

Prunus cerasifera. Tree Physiology 15, 281–93.

Berta G, Fusconi A, Hooker JE, 2002. Arbuscular mycorrhizal

modifications to plant root systems. In: Gianinazzi S, Schuepp H,

Haselwandter K, Barea JM, eds.Mycorrhizal Technology: From

Genes to Bioproducts – Achievement and Hurdles in Arbuscular

Mycorrhizal Research. Basel, Switzerland: ALS Birkhauser

Verlag, 71–101.

Berta G, Sampo S, Gamalero E, Massa N, Lemanceau P, 2005.

Suppression ofRhizoctonia root-rot of tomato byGlomus

mossae BEG12 and Pseudomonas fluorescensA6RI is associated

with their effect on the pathogen growth and on the root

morphogenesis. European Journal of Plant Pathology 111,

279–88.

Conti M, D’Agostino A, Casetta A, Mela L, 1988. Some

characteristics of chrysanthemum yellows disease.Acta

Horticulturae 234, 129–36.

Copetta A, Lingua G, Berta G, 2006. Effects of three AM fungi on

growth, distribution of glandular hairs, and essential oil

production inOcimum basilicum L. var. Genovese.Mycorrhiza

16, 485–94.

D’Amelio R, Massa N, Gamalero E et al., 2007. Preliminary results

on the evaluation of the effects of elicitors of plant resistance on

chrysanthemum yellows phytoplasma infection. Bulletin of

Insectology 60, 317–8.

D’Amelio R, Marzachı C, Bosco D, 2010. Activity of

benzothiadiazole on chrysanthemum yellows phytoplasma

(‘Candidatus Phytoplasma asteris’) infection in daisy plants.

Crop Protection 29, 1094–9.

Fitter AH, 1991. The ecological significance of root system

architecture: an economic approach. In: Atkinson D, ed. Plant

Root Growth, an Ecological Perspective. Oxford, UK: Blackwell

Scientific Publications, 229–43.

Gamalero E, Glick BR, 2011. Mechanisms used by plant growth-

promoting bacteria. In: Maheshwari DK, ed. Bacteria in

Agrobiology: Plant Nutrient Management. Berlin, Germany:

Springer-Verlag, in press. 3

Gamalero E, Martinotti MG, Trotta A, Lemanceau P, Berta G,

2002. Morphogenetic modifications induced by Pseudomonas

fluorescensA6RI andGlomus mosseae BEG12 in the root

system of tomato differ according to plant growth conditions.

New Phytologist 155, 293–300.

Gamalero E, Trotta A, Massa N, Copetta A, Martinotti MG, Berta

G, 2004. Impact of two fluorescent pseudomonads and an

arbuscular mycorrhizal fungus on tomato plant growth, root

architecture and P acquisition.Mycorrhiza 14, 185–92.

Gamalero E, Berta G,Massa N, Glick BR, Lingua G, 2008.

Synergistic interactions between the ACC deaminase-producing

bacterium Pseudomonas putidaUW4 and the AM fungus

Gigaspora rosea positively affect cucumber plant growth. Fems

Microbiology Ecology 64, 459–67.

Gamalero E, Berta G,Massa N, Glick BR, Lingua G, 2010a.

Interactions between Pseudomonas putidaUW4 andGigaspora

rosea BEG9 and their consequences for the growth of cucumber

under salt-stress conditions. Journal of Applied Microbiology

108, 236–45.

Gamalero E, D’Amelio R, Musso C et al., 2010b. Effects of

Pseudomonas putida S1Pf1Rif against chrysanthemum yellows

phytoplasma infection. Phytopathology 8, 805–13.

Gamalero E, Pivato B, Bona E et al., 2010c. Interactions among a

fluorescent pseudomonad, an AM fungus and an hypovirulent

isolate ofRhizoctonia solani affect plant growth and root

architecture of tomato plants. Plant Biosystems 144, 582–91. 4

Garcıa-ChapaM, Batlle A, Lavina A, Camprubı A, Estaun V,

Calvet C, 2004. Tolerance increase to pear decline phytoplasma

in mycorrhizal OF-333 pear rootstock. Acta Horticulturae 657,

437–41.

Guerrieri E, Lingua G, Digilio MC,Massa N, Berta G, 2004. Do

interactions between plant roots and the rhizosphere affect

parasitoid behaviour? Ecological Entomology 29, 753–6.

Hodge A, Berta G, Doussan C,Merchan F, Crespi M, 2009. Plant

root growth, architecture and function. Plant and Soil 321,

153–87.

KaminskaM, Klamkowski K, Berniak H, Sowik I, 2010. Response

of mycorrhizal periwinkle plants to aster yellows phytoplasma

infection.Mycorrhiza 20, 161–6.

Lingua G, D’Agostino G,Massa N, AntosianoM, Berta G, 2002.

Mycorrhiza-induced differential response to a yellows disease in

tomato.Mycorrhiza 12, 191–8.

van Loon LC, Bakker PAHM, Pieterse CMJ, 1998. Systemic

resistance induced by rhizosphere bacteria. Annual Review of

Phytopathology 36, 453–83. 5

Lopez-Raez JA, Verhage A, Fernandez I et al., 2010. Hormonal

and transcriptional profiles highlight common and differential

host responses to arbuscular mycorrhizal fungi and the

regulation of the oxylipin pathway. Journal of Experimental

Botany 61, 2589–601.

MaixnerM, 1994. Transmission of the German grapevine yellows

(Vergilbungskrankheit) by the planthopperHyalesthes obsoletus

(Auchenorryncha: cixiidae).Vitis 33, 103–4.

8 R. D’Amelio et al.

Plant Pathology (2011)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

Marzachi C, Bosco D, 2005. Relative quantification of

chrysanthemum yellows (16Sr I) phytoplasma in its plant and

insect host using real-time polymerase chain reaction.Molecular

Biotechnology 30, 117–27.

Morone C, Boveri M, Giosue S et al., 2007. Epidemiology of

Flavescence doree in vineyards in northwestern Italy.

Phytopathology 97, 1422–7.

Pivato B, Offre P, Marchelli S et al., 2009. Bacterial effects on

arbuscular mycorrhizal fungi and mycorrhiza development as

influenced by the bacteria, fungi, and host plant.Mycorrhiza

19, 81–90.

Ravnskov S, Jakobsen I, 1999. Effects of Pseudomonas fluorescens

DF57 on growth and P uptake of two arbuscular mycorrhizal

fungi in symbiosis with cucumber.Mycorrhiza 8, 329–34.

Romanazzi G, Musetti R, Marzachı C, Casati P, 2009. Induction

of resistance in the control of phytoplasma diseases. Petria 19,

113–29.

Seemuller E, Marcone C, Lauer U, Ragozzino A, Goschl M, 1998.

Current status of molecular classification of the phytoplasmas.

Journal of Plant Pathology 80, 3–26.

Smith SE, Read DJ, 2008.Mycorrhizal Symbiosis. London, UK:

Academic Press.

Trotta A, Varese GC, Gnavi E, Fusconi A, Sampo S, Berta G, 1996.

Interactions between the soilborne root pathogen Phytophthora

nicotianae var. parasitica and the arbuscular mycorrhizal fungus

Glomus mosseae in tomato plants. Plant and Soil 185, 199–

209.

Trouvelot A, Kough JL, Gianinazzi-Pearson V, 1986. Mesure du

taux de mycorhization d’un systeme radiculaire. Recherche de

methodes d’estimation ayant une signification fonctionnelle. In:

Gianinazzi-Pearson V, Gianinazzi S, eds. Physiological and

Genetical Aspects of Mycorrhizae. Dijon, France: INRA,

217–21.

Volante A, Lingua G, Cesaro P et al., 2005. Influence of three

species of arbuscular mycorrhizal fungi on the persistence of

aromatic hydrocarbons in contaminated substrates.Mycorrhiza

16, 43–50.

Weintraub PG, Beanland L, 2006. Insect vectors of phytoplasmas.

Annual Review of Entomology 51, 91–111.

Effects of AM fungus and a pseudomonad on phytoplasmosis 9

Plant Pathology (2011)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

Author Query Form

Journal: PPA

Article: 2479

Dear Author,During the copy-editing of your paper, the following queries arose. Please respond to these by mark-ing up your proofs with the necessary changes/additions. Please write your answers on the query sheetif there is insufficient space on the page proofs. Please write clearly and follow the conventions shownon the attached corrections sheet. If returning the proof by fax do not write too close to the paper’sedge. Please remember that illegible mark-ups may delay publication.

Many thanks for your assistance.

Query reference Query Remarks

1 AUTHOR: Please read through the entire proof carefully, payingparticular attention to the accuracy of equations, tables, illustrations(which may have been redrawn), other numerical matter and refer-ences (which have been corrected for style but not checked for accu-racy, which remains the responsibility of the author).

2 AUTHOR: Please note that the version of your paper that appearsonline is complete and final, except for volume, issue and page num-bers, which are added upon print publication. Therefore, there willbe no further opportunity to make changes to your article afteronline publication.

3 AUTHOR: Please provide the page range for reference Gamalero,Glick (2011).

4 AUTHOR: Gamalero et al. (2010c) has not been cited in the text.Please indicate where it should be cited; or delete from the ReferenceList.

5 AUTHOR: Please check all authors name.

Page 1 of 3

USING E-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION

Required Software

Adobe Acrobat Professional or Acrobat Reader (version 7.0 or above) is required to e-annotate PDFs. Acrobat 8 Reader is a free download: http://www.adobe.com/products/acrobat/readstep2.html

Once you have Acrobat Reader 8 on your PC and open the proof, you will see the Commenting Toolbar (if it does not appear automatically go to Tools>Commenting>Commenting Toolbar). The Commenting Toolbar looks like this:

If you experience problems annotating files in Adobe Acrobat Reader 9 then you may need to change a preference setting in order to edit.

In the “Documents” category under “Edit – Preferences”, please select the category ‘Documents’ and change the setting “PDF/A mode:” to “Never”.

Note Tool — For making notes at specific points in the text

Marks a point on the paper where a note or question needs to be addressed.

Replacement text tool — For deleting one word/section of text and replacing it

Strikes red line through text and opens up a replacement text box.

Cross out text tool — For deleting text when there is nothing to replace selection

Strikes through text in a red line.

How to use it:

1. Right click into area of either inserted text or relevance to note

2. Select Add Note and a yellow speech bubble symbol and text box will appear

3. Type comment into the text box

4. Click the X in the top right hand corner of the note box to close.

How to use it:

1. Select cursor from toolbar

2. Highlight word or sentence

3. Right click

4. Select Replace Text (Comment) option

5. Type replacement text in blue box

6. Click outside of the blue box to close

How to use it:

1. Select cursor from toolbar

2. Highlight word or sentence

3. Right click

4. Select Cross Out Text

Page 2 of 3

Approved tool — For approving a proof and that no corrections at all are required.

Highlight tool — For highlighting selection that should be changed to bold or italic.

Highlights text in yellow and opens up a text box.

Attach File Tool — For inserting large amounts of text or replacement figures as a files.

Inserts symbol and speech bubble where a file has been inserted.

Pencil tool — For circling parts of figures or making freeform marks

Creates freeform shapes with a pencil tool. Particularly with graphics within the proof it may be useful to use the Drawing Markups toolbar. These tools allow you to draw circles, lines and comment on these marks.

How to use it:

1. Click on the Stamp Tool in the toolbar

2. Select the Approved rubber stamp from the ‘standard business’ selection

3. Click on the text where you want to rubber stamp to appear (usually first page)

How to use it:

1. Select Highlighter Tool from the commenting toolbar

2. Highlight the desired text

3. Add a note detailing the required change

How to use it:

1. Select Tools > Drawing Markups > Pencil Tool

2. Draw with the cursor

3. Multiple pieces of pencil annotation can be grouped together

4. Once finished, move the cursor over the shape until an arrowhead appears and right click

5. Select Open Pop-Up Note and type in a details of required change

6. Click the X in the top right hand corner of the note box to close.

How to use it:

1. Click on paperclip icon in the commenting toolbar

2. Click where you want to insert the attachment

3. Select the saved file from your PC/network

4. Select appearance of icon (paperclip, graph, attachment or tag) and close