Natural infectious behavior of the urediniospores of Melampsora larici-populina on poplar leaves

Zhibing Wan?Yiran Li?Min Liu?Yingnan Chen?Tongming Yin

Natural infectious behavior of the urediniospores of Melampsora larici-populina on poplar leaves

Zhibing Wan?Yiran Li?Min Liu?Yingnan Chen?Tongming Yin

The uredinial stage in the life cycle of Melampsora larici-populina on poplar leaves is the most importantpathogenic phase.We captured partialphases of uredinial infection in the wild,aiming to reconstruct the process of uredinial ontogeny by using scanning and transmission electron microscope.At the initial infection stage,germ tubes germinated from the echinulate urediniospores.Germ tubes were frequently seen to merge with the leaf surface and cuticle breakage was observed,indicating direct hyphal penetration.Stomata penetration occurred commonly,sometimes with more than one germ tube penetrating the same stoma.Melampsora larici-populina did not form appressoria in the infection process,implying that infectious behavior of this pathogen may differ from the other rustpathogens.In general,germ tubes branched randomly,and no distinctevidence indicated that stoma could induce or orient germ tube branches.However,oriented germ tube growth has been occasionally observed in otherstudies.The urediniospores collapsed and finally wizened when they became nutrientstressed.Atthe last stage of infection,the uredinia erupted from the leaf epidermis and appeared as orange pustules on the leaf surface.

Populus deltoidesMelampsora laricipopulinaGerm tubeInfectious behavior

Introduction

Populus deltoides naturally occurs in Canada,from eastern Alberta to Quebec,the southern United States,including Florida,Texas,and Arizona,and northern Mexico(Kartesz and Meacham 1999).Populus deltoides isa valuable timber species widely used in short-rotation intensive culture (SRIC)in the United States and Canada(Bull and Muntz 1943).This species has also been introduced as an SRIC worldwide.Populusdeltoides wasintroduced to China in the early 1970s.Presently,this species and its hybrids dominate forestry plantations in areas along the middle and lower reaches of the Yangtze River.However,an outbreak of rust disease has been observed in poplar plantations since the 1990s and is gradually becoming a major problem hampering the developmentofpoplarplantationsin southern China (Wan et al.2013).Based on morphological characteristics and analyses with species-specific molecular markers,the rust pathogens causing these outbreaks were identified as Melampsora larici-populina(Wan etal.2013).

Melampsora larici-populina is the most devastating, widespread pathogen infecting commercially important poplar species(Steenackers etal.1996;Tian etal.1999).It features frequent pathogenic variations and an impressive adaptive potential(McDonald and Linde 2002;Tian et al. 2009).Poplar leaf rustdisease caused by this specific race can lead to more than a 50%reduction in annual growth (Covarellietal.2013).Repeated infections oversuccessive years may resultin the complete loss ofa poplarplantation (Cervera et al.1996).To limit the damage resulting from this pathogen,greatefforts have been made by scientists to develop new breeding strategies.Besides generating a collection of hybrid poplars with complete resistances to M.larici-populina(Newcombe et al.1996;Villar et al. 1996;Lefe′vre etal.1998),poplar breeders have attempted to uncover the host-pathogen interactions(Shain and Jarlfors 1987;Laurans and Pilate 1999;Tian etal.2001)and to identify molecular markers for resistance against M. larici-populina(Cervera et al.1996;Jorge et al.2005). Recently,a combined genomic and transcriptomic approach led to the discovery of a very large repertoire of candidate M.larici-populina effectors(Duplessis et al. 2009,2011;Hacquard et al.2011).Despite considerable efforts by breeders to generate resistant hybrid poplars, rapid breakdown of resistance was generally observed because of the continuous development of new rust pathogens(Shang et al.1986;Newcombe et al.2000; Liang et al.2006).Thus,the sustainability of newly selected resistance-types requires better understanding of the interactions between Populus and M.larici-populina.

In the life cycle of M.larici-populina,the uredialstage on poplar leaves constitutes the most important phase for the pathogen(Mlodzianowski et al.1978).Under favorable conditions,urediniospores form on poplarleavesand rapidly infect neighboring trees.During the infection process,the urediniospores germinate 24 h afterinoculation ofthe poplar (P.deltoides)leaf.After 36 h,haustorium forms,which involves the redirection of the host’s metabolism and the suppression ofhostdefenses(Tian etal.2001).This infection structure is essentialforthe establishmentofa successfulbio trophic relationship(Heath 1997;Voegele et al.2009). Thereafter,a slow expansion of colony size is observed approximately 72 h after inoculation(Tian et al.2001).At approximately 168 h,uredinia erupted from the leaf epidermis and appeared as orange pustules on the leaf surface, leading to anothercycle ofinfection(Hacquard etal.2010). The development of rust disease reduces the assimilation surface of the infected trees(Mlodzianowskietal.1978).

Melampsora larici-populina is a rust pathogen that has recently spread to areas in southern China(Wan et al. 2013).In this study,we aimed to record and reconstructthe process of pathogenesis and the uredinial ontogeny of M. larici-populina on the susceptible host by using scanning and transmission electron microscopy(SEM and TEM, respectively).This work provides information to better understand the Populus–M.larici-populina interaction and verifies the findings of previous studies.

Materials and methods

In mid-October,2011,rust-infected mature leaves of a susceptible P.deltoides,clone‘‘S3503’’,were collected. This clone was imported from Stoneville,Mississippi, USA,in February,1991,and was maintained in the poplar nursery on the campus of Nanjing Forestry University.In the field,this P.deltoides clone was consistently observed to be highly susceptible to M.larici-populina(Wan et al. 2013).

For the SEM examination,urediniospores were airdried,mounted on aluminum stubs and sputter-coated with approximately 30 nm of gold/palladium.The specimens were examined with a JEOL 840A SEM(JEOL,Tokyo, Japan).For the TEM examination,small leaf segments (1 mm 9 2 mm)were fixed in 3%glutaraldehyde and 2%formaldehyde in 0.1 M phosphate buffer(pH 7.2), vacuum infiltrated and stored in the primary fixative at 4C for 24 h after sample collection.Specimens were transferred to fresh buffer(three washes for 30 min)and post-fixed for 30 min to 1 h in 1%osmium tetroxide (OsO4)at4C.Following three further buffer washes,the fixed material was dehydrated in an acetone series(25,50, 75,95,2 9 100%),infiltrated and embedded in Polarbed 812 epoxy resin(cured 72 h at 6C).The grid-mounted sections were stained with saturated uranylacetate in 50% ethanol(3 min),washed in 50%ethanol and distilled water,then stained with lead citrate(3 min)in distilled water.The processed specimens were examined with a Hitachi-600A(Hitachi,Ltd.,Tokyo,Japan)microscope.

Results and discussion

Germ tube development

At the initial stage of symptom development,M.laricipopulina uredinia emerged as yellow-orange pustules of 1–2 mm diameter on the abaxial leaf surface.Then,the uredinia produced a large numberofasexualurediniospores. Under the SEM,the wall surface of the urediniospore appeared echinulate exceptfor a smooth patch atthe broad apex(Fig.1).Germ tubes germinated from the echinulate urediniospores.The wallsofthe hydrated M.larici-populina urediniospores were partially deliquescent,facilitating germination of the germ tubes(Rijkenberg 1972).Germ tubes of M.larici-populina were rope-like in shape and clingywhen appressed to the leaf surface(Fig.2).Spiers and Hopcroft(1988)reported that individual urediniospores contained four germ pores,forming four germ tubes.We found thatone urediniospore generally formed one to three germ tubes butonly occasionally formed four germ tubes.

Fig.1 Detailed morphological characteristics of Melampsora laricipopulina urediniospore

Fig.2 Germ tube sprouting from a Melampsora larici-populina urediniospore

As germ tubes extended,they branched severaltimes to form a dendritic network on the leaf surface(Figs.3,4). Then,afterpenetration ofthe germ tubes,haustoria formed, and the urediniospore became the haustorial mother cell (Spiers and Hopcroft 1988;Tian et al.2002).The cytoplasm of the mother cell migrated continuously into the haustorium,causing the mother cell to become highly vacuolated and itultimately collapsed(Spiers and Hopcroft 1988;Tian et al.2002).In this study,we also observed many collapsed and wizened mother cells(Fig.3).

Fig.3 Wizened and collapsed Melampsora larici-populina urediniospores

Fig.4 The dendritic network formed by Melampsora larici-populina germ tubes on a poplar(Populus deltoids)leaf surface.White arrows indicate the germ tubes merging with each other,and the red arrow indicates the germ tube penetrating the cuticle directly

Direct penetration

Germ tubes varied in morphology when merged with each other,fusing into swollen lizard-or octopus-shaped structures(Figs.4,5).Some germ tubes merged with the epidermis ofthe poplarleaf,and directly penetrated the cuticle (Figs.4,5).Cavities and broken cuticles were found at the infection sites(Fig.5),similar to observations reported by Yu et al.(2011).Spiers and Hopcroft(1988)found a distinguishable hole was made by hyphae through direct penetration;however,no infection structure was formed under the hole.We observed that direct penetration occurred only occasionally.Together with the observation of Spiers and Hopcroft(ibid.),this indicated that directpenetration mightnotbe the main infection path.However, the role of direct penetration remains unclear.Yu et al. (2011)suggested direct penetration of epidermal cells could cause leakage of cell contents,resulting in increased humidity around germ tubes that could reduce the frequency of occurrence of fused germ tubes,which typically formed under low ambient humidity.Additionally, increased humidity could also increase the survivalofgerm tubes in arid environments(Yu et al.2011).

Fig.5 Typical fused cells of germtubes from different Melampsora larici-populina spores.White arrows indicate the germtubes merging with each other and forming a swollen lizard-or octopus-shaped structures.The red arrow indicates the broken poplar(Populus deltoids)leaf cuticle

Fig.6 The Melampsora larici-populina germ tubes passed by the stoma but short branch penetration occurred

Stomatal penetration

Fig.7 Two Melampsora larici-populina germ tubes enter the same stoma

Germ tubes extended on the leaf surface,with clear stomatal invasions,were frequently observed(Figs.6,7), although germ tubes did not take the most direct route to the stomata.They were frequently observed passing over stomata without penetrating(Figs.6,7),but nearby short branches entered opened stomata.The penetration of a single stoma by two germ tubes was commonly observed (Fig.7).Increased magnification revealed that the germ tubes branched randomly(Figs.3,4),and there was no observable indication that germ tubes branched more frequently when near stomata.

In the infection process,the hostsurface appears to play a crucialrole in determining how germ tubes and infection structures develop(Maheshwari et al.1967).Wemer (1982)considered that stomal recognition by M.laricipopulina was dependent on the specific sculpturing of the surface of the stomatal apparatus and the structure of the guard cells.However,no specific orientation of the M. larici-populina germ tubes on the host was observed and they appeared to randomly penetrate stoma(Spiers and Hopcroft1988),like those ofsome otherrusts(Longo etal. 2000;Vaz Patto and Niks 2001;Vaz Patto et al.2009). Whether the leaf surface affects germ tube orientation remains to be determined.Yu et al.(2011)observed that some long germ tubes of M.larici-populina occasionally rolled back near the stomata and formed an infection structure entering the stomata.Thus,more evidence is needed to clarify whether there is an unknown mechanisms triggering stomatal recognition by M.larici-populina.

Evidence from appressorium induction by artificial membranes suggested thatatleasteightrustspecies formed appressoria in response to thigmotropic stimuli(Heath 1997).However,germ tubes were observed to penetrate stomata without forming clearly defined appressoria (Figs.6,7).Chiba(1964),Taris(1968),and Wemer(1982) observed that M.larici-populina did not form appressoria. In a study including Melampsora medusae and M.larici-populina,Spiers and Hopcroft(1988)reported that M. medusae commonly formed appressoria over stomata; however,M.larici-populina did not form appressoria. Since most available studies indicated no appressoria formation during the infection process of M.larici-populina, this species’infectious behaviormay differfrom otherrust pathogens.

Mostof the early studies describing the penetration and establishment of Melampsora rust infection in poplars reported that germ tubes penetrated leaves exclusively via stomata(Chiba 1964;Taris 1968;Mlodzianowski et al. 1978;Wemer 1982).In contrast,Omar and Heather(1976) concluded that the germ tubes of M.larici-populina penetrated leaves directly and very few entered via stomata. Spiers and Hopcroft(1988)observed directpenetration,but this was rare and most germ tubes penetrated leaves via stomata.Yu etal.(2011)observed more directpenetrations compared with those via stomata.Thus,previous reports on the manner of penetration were different,leading to controversy.In this study,we observed both direct and stomatal penetration,with the latter occurring more commonly.The urediniospore germ tube must form the infection structures for infection to occur(Rossetti and Morel 1958;Maheshwari et al.1967).Obvious infection structures were observed after the hyphae penetrated a stoma(Spiers and Hopcroft1988),thus the role ofstomatal penetration was confirmed.As for directpenetration,itdid not result in infection structures(Spiers and Hopcroft 1988),and its role in the infection process remained unclear.

After the hyphae penetrated the leaf tissue,the palisade cells in the leaves of P.deltoides became shrunken and vacuolated(Fig.8).Finally,the uredinia erupted from the leaf epidermis and were seen as orange pustules.In the uredinia,the wall surface of the newly formed urediniospore was smooth atthe initial stage,and became echinulate with the development of urediniospore(Fig.9). Urediniospores were eventually released and disseminated to start another infection process.A complete cycle of infection took about 168 h(Hacquard et al.2010).In a short period of time,the polycylic production of urediniospores occurred,leading to severe epidemics on poplar leaves(Barre`s et al.2008).Uredinia were uniformly distributed on the foliar surface covering the entire leaf blade but did not develop on leaf veins.

Fig.8 The hyphae of Melampsora larici-populina ramifying intercellularly throughout the palisade tissue of Populus deltoids

Fig.9 Newly formed Melampsora larici-populina urediniospores with smooth wall surfaces and an echinulate urediniospore with a smooth patch at the broad apex

Chiba(1964)and Mlodzianowskietal.(1978)examined rust infections(M.larici-populina)on poplars,demonstrating that the host cultivar significantly affected all facets of rust infection,including the rate and extent of urediniospore germination,the extent and pattern of germ tube extension,and whether stomata were penetrated. However,these studies were only performed on a highly susceptible host.Clearly,it is essential to investigate the development of the infection structures of M.larici-populina on poplar cultivars varying in rustresistance.Modern genetics detected the MER locus thatconfers resistance to M.larici-populina(Zhang et al.2001).This locus was mapped to chromosome XIX in the poplar genome(Yin et al.2004).Although the host resistance to rust can be resolved at the gene level,understanding the infectious behavior of ruston the leaf surface is criticalin elucidating pathogen-host interactions.

Barre`s B,Halkett F,Dutech C,Andrieux A,Pinon J,Frey P(2008) Genetic structure of the poplar rust fungus Melampsora laricipopulina:evidence for isolation by distance in Europe and recent founder effects overseas.Infect Genet Evol 8:577–587

Bull H,Muntz HH.1943.Planting cottonwood on bottomlands. Bulletin 391.Mississippi State,MS:Mississippi State College, Agricultural Experiment Station.18 p

Cervera MT,Gusmao J,Steenackers M,Gysel AV,Montagu MV, Boerjan W(1996)Application of AFLPTM-based molecular markers to breeding of Populus spp.Plant Growth Regul 20:41–52

Chiba O(1964)Studies on the variation in susceptibility and the nature of resistance of poplars to the leaf rust caused by Melampsora larici-populina Klebahn.Bull Gov For Exp Stn Tokyo 166:157

Covarelli L,Beccari G,Tosi L,Fabre B,Frey P(2013)Three-year investigations on leafrustofpoplarcultivated forbiomass production in Umbria,CentralItaly.Biomass Bioenergy 49:315–322

Duplessis S,Major I,Martin F,Se′e′guin A(2009)Poplar and pathogen interactions:insights from Populus genome-wide analyses of resistance and defense gene families and gene expression profiling.Crit Rev Plant Sci 28:309–334

Duplessis S,Hacquard S,Delaruelle C,TisserantE,Frey P,Martin F, Kohler A(2011)Melampsora larici-populina transcriptprofiling during germination and time course infection of poplar leaves reveals dynamic expression patterns associated with virulence and biotrophy.Mol Plant Microbe Interact24:808–818

Hacquard S,Delaruelle C,Legue′V,Tisserant E,Kohler A,Frey P, Martin F,Duplessis S(2010)Laser capture microdissection of uredinia formed by Melampsora larici-populina revealed a transcriptional switch between biotrophy and sporulation.Mol Plant Microbe Interact 23:1275–1286

Hacquard S,Petre B,Frey P,Hecker A,Rouhier N,Duplessis S (2011)The poplar-poplar rust interaction:insights from genomics and transcriptomics.J Pathog 2011:1–11

Heath MC(1997)Signalling between pathogenic rust fungi and resistant or susceptible host plants.Ann Bot 80:713–720

Jorge V,Dowkiw A,Faivre-Rampant P,Bastien C(2005)Genetic architecture of qualitative and quantitative Melampsora laricipopulin a leaf rust resistance in hybrid poplar:genetic mapping and QTL detection.New Phytol 167:113–127

Kartesz JT,Meacham CA(1999)Synthesis of the North American flora(Windows Version 1.0),[CD-ROM].In:North Carolina botanical garden,Cooperation with the nature resources conservation Service,and U.S.Fish and wildlife service,Chapel Hill

Laurans F,Pilate G(1999)Histological aspects of a hypersensitive response in poplar to Melampsora larici-populina.Phytopathology 89:233–238

Lefe′vre F,Goue′-Mourier MC,Faivre-Rampant P,Villar M(1998)A single gene cluster controls incompatibility and partialresistance to various Melampsora larici-populina races in hybrid poplars. Phytopathology 88:156–163

Liang Y,Tian C,Kakishima M(2006)A new species of Melampsora on Populus yunnanensis from China.Mycoscience 47:198–204

Longo N,Poggiolesi S,Naldini B,Tani G(2000)Penetration and early colonization in basidiosporederived infection on needles of Pinus pinea L.by Cronartium flaccidum(Alb.et Schw.)Wint. Caryologia 53:9–29

Maheshwari R,Allen PJ,Hildebrandt AC(1967)Physical and chemical factors controlling the development of infection structures from urediniospore germ tubes of rust fungi.Phytopathology 57:855–862

McDonald B,Linde C(2002)Pathogen population genetics,evolutionary potential,and durable resistance.Annu Rev Phytopathol 40:349–379

Mlodzianowski F,Werner A,Siwecki R(1978)Germination of Melampsora larici-populina urediniospores on poplar leaves. Eur J For Pathol 8:119–125

Newcombe G,Bradshaw HD Jr,Chastagner GA,Stettler RF(1996)A major gene for resistance to Melampsora medusae f.sp. deltoidae in a hybrid poplar pedigree.Phytopathology 86:87–94

Newcombe G,Stirling B,McDonald S,Bradshaw HD Jr(2000) Melampsora 9 columbiana,a naturalhybrid of M.medusae and M.occidentalis.Mycol Res 104:261–274

Omar MB,Heather WA(1976)Scanning electron microscope studies on the behaviour of urediniospores of Melampsora laricipopulina.In:Proceedings of XIX session FAO/IPC working party on poplar diseases,France

Rijkenberg FHJ(1972)Fine structure of the poplar rust(Melampsora larici-populina).Phytophylactica 4:33–40

Rossetti V,Morel G(1958)Le developpementdu Puccinia antirrhini sur tissue du Muflier cultives in vitro.Comptes rendus Hebd sdances Acad Sci Ser D 247:1893–1895

Shain L,Jarlfors U(1987)Ultrastructure of eastern cottonwood clones susceptible or resistant to leaf rust.Can J Bot 65:1586–1598

Shang YZ,Pei MH,Yuan ZW(1986)A new rustfungus on poplars. Acta Mycol Sin Suppl 1:180–184(in Chinese)

Spiers AG,Hopcroft DH(1988)Penetration and infection of poplar leaves by urediniospores of Melampsora laricipopulina and Melampsora medusae.NZ J Bot 26:101–111

Steenackers J,Steenackers M,Steenackers V,Stevens M(1996) Poplar diseases,consequences on growth and wood quality. Biomass Bioenergy 10:267–274

Taris B(1968)Contribution a l’e′tude des rouilles des Populus observe′es en France.Ann Epiphyt 19:5–54

Tian C,LiZ,Kang Z(1999)Advances in researches ofcathay poplar leaf rust(Melampsora larici-populina Kleb.).J Northwest For Univ 14:81–88(in Chinese)

Tian C,Liang Y,Kang Z,LiZ,Zhao Y(2001)Histopathology studies on interaction of poplars and leaf rust with different compatibilities.Sci Silvae Sin 37:52–58(in Chinese)

Tian C,Liang Y,Kang Z,Li Z,Zhao Y(2002)Ultrastructure of poplar leaf infected by rustfungus(Melampsora larici-populina Kleb.).Acta Phytopathol Sin 32:71–78(in Chinese)

Tian C,Zhao P,Cao Z(2009)Role of cellwalldegrading enzymes in the interaction of poplar and Melampsora larici-populina Kleb. Front For China 4:111–116

Vaz Patto MC,Niks RE(2001)Leaf wax layer may prevent appressorium differentiation butdoes notinfluence orientation of the leafrustfungus Puccinia hordei on Hordeum chilense leaves. Eur J Plant Pathol 107:795–803

Vaz Patto MC,Fernandez-Aparicio M,Moral A,Rubiales D(2009) Pre and posthaustorial resistance to rusts in Lathyrus cicera L. Euphytica 165:27–34

Villar M,Lefe′vre F,Bradshaw HD,Tessier du Cros E(1996) Molecular genetics of rust resistance in poplars(Melampsora laricipopulina Kleb/Populus sp.)by bulked segregantanalysis in a 2 9 2 factorial mating design.Genetics 143:531–536

Voegele RT,Hahn M,Mendgen K(2009)The uredinales:cytology, biochemistry,and molecular biology.In:Deising HB(ed)The mycota,5,vol 2.,Plant relationshipsSpringer,Berlin,pp 69–98

Wan ZB,Li YR,Chen YN,Zhang XY,Guan HW,Yin TM(2013) Melampsora larici-populina,the main rustpathogen,causes loss in biomass production of black cottonwood plantations in the south of China.Phytoparasitica 41:337–344

Wemer A(1982)Histological studies on the host-pathogen relationships in the course of disease development caused by Melampsora larici-populina Kleb.Arbor Korn 26:51–99

Yin TM,DiFazio SP,Gunter LE,Jawdy SS,Boerjan W,Tuskan GA (2004)Genetic and physical mapping of Melampsora rust resistance genes in Populus and characterization of linkage disequilibrium and flanking genomic sequence.New Phytol 164:95–105

Yu ZD,Peng SB,Ren ZZ,Wang DM,Cao ZM(2011)Infection behaviour of Melampsora larici-populina on the leaf surface of Populus purdomii.Agric Sci China 10:1562–1569

Zhang J,Steenackers M,Storme V,Neyrinck S,Van Montagu M, Gerats T,Boerjan W(2001)Fine mapping and identification of nucleotide binding site/leucine-rich repeatsequences atthe MER locus in Populus deltoides‘s9-2’.Phytopathology 91:1069–1073

19 September 2013/Accepted:24 January 2014/Published online:21 January 2015

Project funding:This work was financially supported by the Key Forestry Public Welfare Project(201304102),the National Natural Science Foundation(No.31400563),the Key Project of Jiangsu Foundation of Natural Sciences for High Education(10KJA180018) and the Natural Science Foundation of Jiangsu Province (BK20130968).This work was also supported by the program for Innovative Research Teams in the Universities of Jiangsu Province and the Educational Departmentof China,and the Priority Academic Program Development(PAPD)of Jiangsu Higher Education Institutions.

The online version is available at http://www.springerlink.com

Corresponding editor:Hu Yanbo

Z.Wan and Y.Li contributed equally to this research.

The Southern Modern Forestry Collaborative Innovation Center, Nanjing Forestry University,Nanjing 210037,China

e-mail:chenyingnan@njfu.edu.cn

Z.Wan

Schoolof Life and Environment Science,Huangshan University, Huangshan 245041,China