Gall production on hawthorns caused by Gymnosporangiumspp. in Hatay province, Turkey

Sibel Dervis & Linley Dixon & Mikdat Doğanlar &

Amy Rossman

Received: 26 October 2009 /Accepted: 25 May 2010 /Published online: 10 June 2010# Springer Science+Business Media B.V. 2010

Abstract Three hawthorn and related rust diseasescaused by Gymnosporangium confusum on Crataegusmonogyna, Gymnosporangium clavariiforme onCrataegus orientalis and Gymnosporangium sabinaeon Pyrus communis were detected in Hatay province,Turkey. G. confusum was also found causing telialgalls on Juniperus communis. G. confusum and G.clavariiforme produced aecial horns on overwinteredgalls on hawthorn twigs from May to June. Theproduction of galls caused by Gymnosporangium onthe secondary host is unusual. Portions of the nuclearITS and LSU rDNA were sequenced for all threespecies of Gymnosporangium for use as a speciesbarcode; sequences were deposited in GenBank.Sequence data from G. clavariiforme and G. sabinaematched those in GenBank; however, this is the firststudy to deposit sequence data from G. confusum toGenBank. The life cycles of G. confusum and G.

clavariiforme are discussed along with implicationsfor disease control.

Keywords Crataegus .Gymnosporangiumconfusum .Gymnosporangium clavariiforme .

Overwintering aecia

Introduction

Species of Gymnosporangium are major pathogenicrust fungi causing diseases and significant economiclosses to plants in the Cupressaceae and Rosaceae(Kern 1973; Parmelee 1965, 1971; Sinclair and Lyon2005). These species are known to delay harvest ofrosaceous fruits by inhibiting photosynthesis andincreasing respiration rates (Kim and Kim 1980).About 60 species of Gymnosporangium with 15species of Roestelia as anamorphs are known inPaleartic regions (Kern 1973; Wilson and Henderson1966). Recently, Yun et al. (2009) described two newspecies of Gymnosporangium from Korea distin-guished by telial characteristics and LSU rDNAsequences. From Turkey, seven species of Gymno-sporangium have been reported to date: G. amelan-chieris E. Fisch. ex F. Kern, G. clavariiforme(Wulfen) DC., G. confusum Plowr., G. cornutumArthur ex F. Kern, G. dobrozrakovae Mitrof., G.sabinae (Dicks.) G. Winter (as G. fuscum DC.), andG. tremelloides R. Hartig (Bahçecioğlu 2001; Dinç

Phytoparasitica (2010) 38:391–400DOI 10.1007/s12600-010-0102-z

S. Dervis (*) :M. DoğanlarDepartment of Plant Protection, Faculty of Agriculture,University of Mustafa Kemal,31034 Antakya, Hatay, Turkeye-mail: sdervis@mku.edu.tr

L. Dixon :A. RossmanUnited States Department of Agriculture,Systematic Mycology & Microbiology Laboratory,Agricultural Research Service,Beltsville, MD 20705, USA

Tab

le1

The

occurrence

ofGym

nosporan

gium

confusum

,G.clavariiformeandG.sabina

ein

Hatay

prov

ince

Sam

plingtim

eHostsandmaterialssampled

No.

ofsamples

/15

Crataegus

mon

ogyna

trees(w

ithgalls)

No.

ofsamples

/15

Crataegus

mon

ogynatrees

(with

outgalls)

No.

ofsamples

/20

Crataegus

orientalistrees

No.

ofsamples

/20

Pyrus

commun

istrees

No.

ofsamples

/25

Juniperusspp.

trees

Patho

gen:

G.confusum

G.clavariiforme

G.sabina

eG.confusum

/G.sabina

e

February

37galls

–5galls

––

1March

15galls

–7galls

––

8March

5galls

+25

infected

flow

ers

5infected

leaves

Noinfected

leaves

orflow

ers

––

15March

100infected

leaves

&flow

ers

5infected

leaves

3infected

flow

ers

Bud

sdeveloped

––

22March

90%

ofleaves

&flow

ersinfected

30%

ofleaves

&flow

ersand

10%

offruitsinfected

4infected

leaves

––

7&

14April

Infected

partswith

aecia

(abo

ut10

0specim

ens)

Sam

e(abo

ut25

specim

ens)

15infected

leaves

––

21&

28April

Gallform

ations

startedon

shoo

tsandtwigs

Sam

eSom

eof

theleaves

with

aecia

15leaves

with

spots

5galls

swelledon

branches

May

Horndevelopm

entstartedon

galls

Sam

eInfected

partshaving

aecia

&aeciospo

reson

both

sides

ofleaves,new

gallform

ations

ontwigs

Leaveswith

yello

wspotson

upperside

andbrow

nish

smallspotson

undersides

Gallswith

cracks

onbranches

June

Som

ebrok

enho

rns,many

aeciospo

reswerespread

arou

ndSam

eHorndevelopm

ent,abno

rmally

twistedleaves

&fruits

Num

berof

infected

leaves

andfruitsincreased

Gallswith

sameappearance

392 Phytoparasitica (2010) 38:391–400

and Yilmaz 1978; Erdoğdu et al. 2010; Hüseyinov2000; Hüseyinov and Selçuk 2001).

In general, rust fungi in the genus Gymnosporan-gium alternate their development between primaryhosts in the Cupressaceae mainly on Cupressus andJuniperus and secondary hosts in the Rosaceae onCrataegus, Malus, Pyrus and Sorbus (Kern 1973;Peterson 1982; Sinclair and Lyon 2005; Ziller 1974).Sinclair and Lyon (2005) described the life cycle ofGymnosporangium as having four to five differentspore stages, lacking a uredinial stage except for G.gaeumannii H. Zogg, G. nootkatense Arthur, and G.paraphysatum Vienn.-Bourg., with infection on theaecial host limited to one growing season. Infection onCupressaceae is initiated by aeciospores, which developinto teliospore-producing galls causing stem swellings,witches’ brooms, and dieback of twigs and branches.

Teliospores germinate to produce basidiospores, whichinfect rosaceous hosts. Symptoms on rosaceous hostsinclude pigmented leaf spots and swellings localized onleaves, fruits and green twigs, which are caused by theproduction of spermatia and aeciospores. Infections onthe secondary host are often followed by casting ordistortion and death of these plant parts. The productionof galls caused by Gymnosporangium species on thesecondary host is unusual.

Several species of Gymnosporangium are commonlyreported on Crataegus, or hawthorns, throughout theworld. In Turkey only two species have been reportedon hawthorn: G. clavariiforme (Dinç and Yilmaz 1978;Karaca 1965) and G. confusum (Kabaktepe andBahçelioğlu 2006; Karaca 1965). Dinç and Yilmaz(1978) reported that G. confusum was restricted toquince (Cydonia oblonga Mill.) orchards in Tanırvillage, Kahramanmaraş province, Şüşnaz and Harput(Elazığ) and Tunceli, although G. confusum hasrecently been reported on Crataegus microphylla K.Koch in Ordu (Kabaktepe and Bahçelioğlu 2006). Inthe same study, G. clavariiforme was found on Pyrusspp. in Gölbaşı village (Doğanşehir-Malatya) and onCrateagus spp. in Bahçe (Adana), Tellidere Village(İskenderun-Hatay), and Arapgir (Osmanpaşa-Malatya).G. clavariiforme has also been reported on Crataegusmonogyna Jacq. and Crataegus orientalis Pall. ex M.Bieb. (Karaca 1965). Both G. clavariiforme and G.confusum were also reported on Crataegus spp. inSivas (Bahçelioğlu and Yildiz 2005) and in theMediterranean parts of Europe in the 1950’s (Bernaux1956).

Hawthorn (Crateagus spp.) is a native plant of theMediterranean regions of North Africa, Europe, and

Fig. 1 Overwintering galls of Gymnosporangium confusum onCrataegus monogyna containing aeciospores

Fig. 2 Aeciospores ofGymnosporangiumconfusum formed in aeciaon the lower leaf surface ofCrataegus monogyna and instromatic structures inoverwintered galls showingsurface ornamentation

Phytoparasitica (2010) 38:391–400 393

central Asia, and now grows in many areas of NorthAmerica. Turkey is one of the centers for geneticdiversity of Crataegus (Ercisli 2004). Recently, haw-thorns have been cultivated as a horticultural crop for

their fruits and also for medicinal purposes in severalparts of Turkey and distributed to other provinces. Itis reported that hawthorn fruits have high flavonoid,vitamin C, glycoside, anthocyanin, saponin, tannin,

Fig. 3 Life cycle of Gymnosporangium confusum in Hatay province

Fig. 4 Infections onleaves (a), flowers (b) andfruits (c) of Crataegusmonogyna caused byGymnosporangiumconfusum

394 Phytoparasitica (2010) 38:391–400

and antioxidant levels (Ljubuncic et al. 2005) as wellas phenolics (Chang and Zuo 2002; Schussler andHolzl 1995).

The objectives of this study were to (i) identifyhawthorn rusts in the orchards and forest areas inHatay province, Turkey, based on morphology andanalyses of DNA sequence data; and (ii) documentthe rust life cycles for disease control.

Materials and methods

Morphological identification Fruit orchards and forestsin Altınözü, Antakya, Belen, Samandağ, andYayladağı districts of Hatay province were surveyedfrom 2007 to 2009. Leaf lesions and branches withgalls from Crataegus monogyna, C. orientalis, Cydo-nia oblonga, Malus domestica L., Pyrus communis L.(Rosaceae), and Juniperus spp. (Cupressaceae) werecollected and brought to the laboratory in paper bagsbetween February and August each year. In 2007 and2008, only galls were sampled. Tissues of infectedparts were boiled in 10% KOH solution for about5 min and slides were prepared for morphologicalidentification of the causal agent. Then 30, 20, 20 and25 trees of C. monogynya, C. orientalis, P. communisand Juniperus spp., respectively, were selected fromrust infection-positive areas in all locations. Thespecimens from those trees were coded and labeledbased on the sampling district and tree. Afteridentification, records from field and laboratoryobservations were gathered for the respective species.Therefore, the occurrence of each species wasdetermined with the life stage of the pathogen oninfected plant parts on each date surveyed; these dataare presented in Table 1.

Molecular identification Tissue with rust symptomsfrom C. monogyna, C. orientalis, J. communis, andP. communis were sent to the USDA SystematicMycology & Microbiology Laboratory in Beltsville,

Fig. 5 Spermogonium of Gymnosporangium confusum in thecenter of lesions on the upper leaf surface of Crataegusmonogyna

Fig. 6 Aecial cups of Gymnosporangium confusum on lowerleaf surface of Crataegus monogyna (Scale bar=2 mm)

Fig. 7 Aecial horns of Gymnosporangium confusum protrud-ing from galls on twigs of Crataegus monogyna

Phytoparasitica (2010) 38:391–400 395

MD, for molecular identification. A small amount ofsymptomatic tissue from each sample was added tobead solution tubes from the Mo Bio UltraClean PlantDNA extraction kit (Carlsbad, CA, USA). Samples

were processed twice in a FastPrep (Bio101, Vista,CA, USA) for 30 s at 5.0 m s−1 with 10-min room-temperature incubations between runs. Genomic DNAwas extracted according to the manufacturer’s spec-

Fig. 8 Gall formation on Crataegus monogyna caused by Gymnosporangium confusum. (a) Aecia forming gall on shoot; (b) Springgall with aecial horns; (c) Aecial gall in winter; (d) Horizontal section of overwintered galls

Fig. 9 Overwintered galls of Gymnosporangium confusumforming aecial horns on twigs of Crataegus monogyna (Scalebar=2 mm)

Fig. 10 Two-celled teliospores of Gymnosporangium confu-sum inside galls on Juniperus communis (Scale bar=10 µm)

396 Phytoparasitica (2010) 38:391–400

ifications except that tubes were incubated at 55°Covernight in P1 solution. Primer pairs Rust2inv (Aime2006) and LR6 (Vilgalys and Hester 1990) were usedto PCR (polymerase chain reaction) portions of theITS and nLSU gene regions and products weresequenced with an ABI 3130 (Applied Biosystems,Foster City, CA, USA) using the PCR primers andLR3 (Vilgalys and Hester 1990) and LROR(Moncalvo et al. 1995). Sequences were assembledand edited using Sequencher 4.8 (Gene Codes,

Madison, WI, USA) and compared with those fromGymnosporangium species in GenBank.

Results

Based on morphological and microscopic analysis ofrDNA sequence data, three species were identified,

Fig. 11 Leaf spots of Gymnosporangium clavariiforme on Crataegus orientalis with spermogonia (lower left) and aecial cups on bothupper and lower leaf surfaces (Scale bar=2.5 mm)

Fig. 12 Aecial hornsof Gymnosporangiumclavariiforme on Crataegusorientalis

Phytoparasitica (2010) 38:391–400 397

namely, G. clavariiforme on C. orientalis, G. confusumon C. monogyna, and G. sabinae on P. communis. G.confusum was also found on J. communis. Othermembers of the Rosaceae, including M. domesticaand C. oblonga, were not found to be infected byspecies of Gymnosporangium. Sequences from the ITSregion and the 28S ribosomal RNA gene were depositedin GenBank: G. confusum (GU058011, HM114219), G.clavariiforme (HM114220), G. sabinae (HM114221).DNA sequence data of the Gymnosporangium rustcollected on C. monogyna matched the Gymnospor-angium rust from Juniperus spp. This rust wasidentified as G. confusum based only on morpholo-gy, since there were no G. confusum sequences onGenBank, prior to this collection. Morphologicalfeatures of the Gymnosporangium rust from C.orientalis were consistent with G. clavariiformeand two point mutations separate this isolate fromthe G. clavari i forme i sola te in GenBank(AF426211). Morphological features of the Gymno-sporangium rust from P. communis were consistentwith G. sabinae and two point mutations separatethis isolate from the two G. sabinae sequences inGenBank (AY512845, AF426209).

Spore formations and types of infection in Gymno-sporangium spp.Gymnosporangium confusum In February, somegall formations were observed on C. monogyna(wild hawthorn) (Fig. 1). In the first half of March(Table 1), leaf and flower spots caused by G.confusum were observed on the same host. Uponmicroscopic examination, aecia containing aecio-spores were found on lower leaf surfaces and in galls(Fig. 2). Primary infections were caused by basidio-spores released from galls on J. communis (Fig. 3) in thefirst week of April. Infections occurred mainly on newlydeveloped leaves, flowers and fruits (Fig. 4) of C.monogyna. Symptoms developed on the upper sidesof leaves; then spermogonia with few spermatiadeveloped in the center of these lesions (Fig. 5).Aecia developed on the underside of these lesions.In April, aecial peridia developed and aeciosporeswere released to potentially infect J. communis(Fig. 6). Aecial horns lacking a peridium began todevelop in the beginning of May and were fullyformed and releasing aeciospores in approximately10 days (Fig. 7). Galls containing aecia on haw-thorns apparently overwintered (Figs. 8 and 9).

In May–June, new galls were detected on J.communis containing few teliospores but lacking horndevelopment. Junipers with small, thin galls werefound on nearby wild and cultivated hawthorns andapple trees. Galls containing telia had tongue-shapedgall cracks (Fig. 3), although teliospores were sparse(Fig. 10). Teliospores were two-celled, ellipsoid,brownish orange, and 38–46×25–34 µm. RibosomalDNA sequences from these galls matched thoseisolated from C. monogyna.

Gymnosporangium clavariiforme Infections causedby G. clavariiforme were not observed until thesecond half of April on C. orientalis. Primary leafinfections were caused by basidiospores released fromneighboring junipers. Infections occurred mainly onnewly developed leaves and flowers of C. orientalis.Symptoms were observed on both upper and lowerleaf surfaces, although aecia were more prominent onlower leaf surfaces (Fig. 11). In May, gall formationoccurred on C. orientalis from infections initiated inApril. In early June, horns began developing on thesegalls, and aecia were released in approximately 10–15 days, potentially causing infections on J. communis(Fig. 12).

Gymnosporangium sabinae In all surveyed areas, thefirst disease symptoms on P. communis were caused bythe spermogonial stage of G. sabinae and were observedin late April. The aecial stage did not form until October.Infections of P. communis by G. sabinae were observedabout 2 months later than those observed on hawthorn,caused by G. communis and G. clavariiforme.

Discussion

Sequence data from the nuclear ribosomal ITS2 and the5′ portion of the LSU locus were useful as barcodes foridentifying G. clavariiforme and G. communis tospecies. Identification of G. confusum was based onsequence data (M. C. Aime, personal communication)and morphology since there were no G. confusumsequences deposited to GenBank prior to this study.

Gymnosporangium clavariiforme and G. confusumproduced overwintered aecial galls on their respec-tive secondary hosts, namely, C. orientalis and C.monogyna. Aecia were produced within theseoverwintered galls from April to June. Additional

398 Phytoparasitica (2010) 38:391–400

infections were observed in May on fruits and leavesof Crataegus, presumably caused by the additionalrelease of basidiospores from Juniperus spp.

The absence of teliospore horns on junipers in thisstudy differs from previous reports describing numer-ous teliospore horns on ornamental juniper plants andmajor rust epidemics in Turkey and Europe (Erdoğduet al. 2010; Kern 1973; Peterson 1982). Only fewteliospores were found inside gall cracks on juniperbranches, although one would expect to find telialhorns abundantly in May–October.

Although G. confusum and G. clavariiforme havebeen reported to occur on apple and quince (Dinç andYilmaz 1978), we did not find rust symptoms on thesespecies. The known host range of G. confusumincludes Cotoneaster spp., Crataegus spp., Cydoniaspp., Eriobotrya japonica (Thunb.) Lindl., Juniperusspp., Malus spp., Pyracantha coccinea M. Roem.,Pyrus spp., and Sorbus spp. The known host range ofG. clavariiforme includes Amelanchier spp., Aroniaarbutifolia (L.) Pers., Cotoneaster spp., × Crataego-mespilus spp. (Crataegus × Mespilus), Crataegus spp.,Cydonia spp., Juniperus spp., Malus sylvestris (L.)Mill., Pyrus spp., and Sorbus spp. (Farr and Rossman2010). These hosts include widely distributedornamentals and indigenous plants in areas surround-ing the cultivation of rosaceous plants. Since their hostranges overlap, diagnostic features distinguishing thetwo common species of Gymnosporangium of Cratae-gus in Turkey are important for accurate identification.An understanding of the life cycles of these fungi mayhelp control diseases caused by Gymnosporangium oneconomically important Rosaceae.

In 2007 and 2008 one species of megastigminewasp, Westalianus altinozus Doğanlar (Hymenoptera,Torymidae), one Sinoxylon sp. (Coleoptera, Bostry-chidae), and some unidentified lepidopterous larvaewere found feeding inside the galls on C. monogyna.Westalianus altinozus was found as a new parasiticspecies (Doğanlar 2010) on the caterpillars ofMicrolepidoptera. Sinoxylon spp. move between treesfor feeding and likely carry fungal spores with them.Gall-inhabiting insects may play a role in sporedispersal of G. clavariiforme and G. confusum inHatay. In areas with a high natural presence of bothGymnosporangium species, the sanitation of galls onhawthorns is strongly recommended for diseasecontrol before the initiation of primary infection andthe development of aecial horns.

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