Research Institute of Pomology and Floriculture, Skierniewice, Poland

EXPERIMENTAL TRANSMISSION OF PHYTOPLASMASFROM DISEASED MAGNOLIAS TO CATHARANTHUS ROSEUS

TEST PLANTS BY GRAFTING

H. Śliwa and M. Kamińska

Abstract

Phytoplasmas classified as aster yellows and apple proliferation phytoplasmaswere transmitted from naturally infected magnolias with stunting disease symp-toms to periwinkle (Catharanthus roseus) by grafting. The identity of the graft-trans-mitted phytoplasmas was confirmed by RFLP analysis of polymerase chainreaction (PCR) amplified 16S rDNA fragments.

Key words: magnolia, stunting disease, phytoplasma, PCR, RFLP, graft transmis-sion

Introduction

Magnolias are relatively free of pest and disease problems, however a new se-vere disease was recorded recently in Magnolia spp. plants (Kamińska et al. 2001b). The symptoms included stunted growth, leaf necrosis and malformation, apicalshoot necrosis and abnormal production of secondary shoots. The disease occursin some gardens and nurseries in Poland and in the imported plants. Despite nega-tive results of electron microscopy examination, the data obtained by PCR amplifi-cation of 16S rDNA and RFLP analysis revealed for the first time that incidence ofthe symptoms in magnolia plants was associated with infection of the aster yellowsphytoplasma (16SrI) and a phytoplasma related to apple proliferation phytoplasmagroup (16SrX) (Kamińska and Śliwa 2003). The authors’ study showed also thatantibiotic treatments temporary promoted shoot growth and the development ofasymptomatic leaves and flower buds of naturally infected magnolias, thereby sup-porting the idea that magnolia stunt is caused by phytoplasmas.

Phytopathol. Pol. 32: 21–31© The Polish Phytopathological Society, Poznań 2004ISSN 1230-0462

Phytoplasmas are known to cause diseases in several hundred plants species(Plant diseases… 1989, Seemüller et al. 1998 a). Many of them, including aster yel-lows phytoplasma group (AY; 16SrI) and apple proliferation phytoplasma group(AP; 16SrX) are of considerable economic importance. The phytoplasmas calledaster yellows are associated with a range of diseases in Europe and America andthey are transmitted by leafhoppers (Lee et al. 1993, Vibio et al. 1996). In Poland,they were identified in several ornamental crops (Kamińska and Korbin 2002,Kamińska et al. 1997, 2001 a, b).

According to the current molecular classification scheme (Lee et al. 1998,Seemüller et al. 1998 a), the phytoplasmas responsible for apple proliferation (AP;16SrX-A), European stone fruit yellows (ESFY; 16SrX-B) and pear decline diseases(PD; 16SrX-C) of fruit trees in Europe, are genetically related and they belong tothe phylogenetic group of apple proliferation. RFLP profiles corresponding to APproliferation phytoplasma were found in European hazel (Marcone et al. 1996),Celtis australis L. (Bertaccini et al. 1996), Spartium junceum L., Sarothamnus scoparius(L.) Winm. (Marcone et al. 1997 b, c) and peach trees in USA (Scott and Zimmer-man 2001). Very recent publications provided evidence that a mixed infection of16SrI and 16SrX was detected in fruit trees (Lee et al. 1995, Paltrinieri et al. 2001,Bertaccini et al. 2001) and in red and white current with full blossom symptoms inCzech Republic (Navrátil et al. 2001). Phytoplasmas belonging to AP group aretransmitted by psyllids, which overwinter as infectious adults (Carraro et al. 2001,Frisinghelli et al. 2000). In Poland, apple proliferation disease has been knownfrom more than 30 years (Kamińska and Zawadzka 1970) and pear decline wasfirst identified in 1996 (Malinowski et al. 1996).

Work with phytoplasmas of woody plants is difficult due to low titre and sea-sonal fluctuation of these pathogens. Some of these problems can be solved bytransmission phytoplasmas to periwinkle (Catharanthus roseus L.G. Don). Periwin-kle, commonly used as a source plant to maintain phytoplasma isolates, can har-bour the majority of known phytoplasmas, and greatly facilitate the research.Several phytoplasmas causing important diseases of trees, e.g. white ash declining(Hiben and Wolanski 1971) and alder yellows (Marcone et al. 1997 a) have beentransmitted to periwinkle using Cuscuta spp. plant, while apple proliferationphytoplasma – by psyllids (Refatti et al. 1986) and by dodder (Carraro et al. 1988,Loi et al. 1995, Marcone et al. 1999). Some aster yellows phytoplasma isolates havebeen transmitted to periwinkle by grafting (Kamińska and Korbin 1999, Kamińskaet al. 1997, 2001 a).

The main objectives of this work were to confirm the association of an AY andAP phytoplasmas with magnolia stunting disease by PCR with universal and groupspecific phytoplasma primers and RFLP analyses, and to study the transmissionrate of phytoplasmas through grafting with buds collected from diseased magno-lias to C. roseus test plants.

22 H. Śliwa and M. Kamińska

Materials and methods

Plant material

Nine two years old Magnolia liliiflora L. cv. ‘Nigra’ plants were collected in acommercial nursery in central Poland. In the nursery they were propagated bywoody cuttings in summer and cultivated in a greenhouse. All plants showedstunted growth, leaf necrosis and malformation, shoot proliferation and dieback.

Graft-transmission

Seven or eight periwinkle seedlings, in the age of three–four months, weregrafted each with two bud chips from each selected donor plant. Buds for graftingwere collected in June or August 2001. For comparison five plants were left notgrafted. The experimentally inoculated plants were maintained in an insect-proofgreenhouse and observed for the development of symptoms such as leaf discolor-ation and diminishing, poor growth, secondary shoot production and flower ab-normalities for two growing seasons. In order to verify that the disease symptomswere due to phytoplasmal infection, about half of the plants were examined for thepresence of the phytoplasma by PCR.

Plant samples and the reference strains

For each of the magnolia plant samples of tissue from the growing shoots weretaken in May and August 2001 or October 2002. Leaf samples were collected from29 experimentally inoculated by grafting C. roseus plants. The first samples werecollected three–four months after inoculation; the final testing was made in No-vember 2002.

For comparison leaf samples from plants inoculated with the reference strain ofaster yellows phytoplasma (AY1, 16SrI-B, kindly supplied by Dr. I.-M. Lee,Beltsville, USA) and apple proliferation phytoplasma (AP; 16SrX-A, kindly sup-plied by Dr. A. Bertaccini, Bologna, Italy) were included.

Phytoplasma detection and identification

DNA was isolated from approximately 1 g of fresh tissue (leaves or phloemstem) using the phytoplasma enrichment procedure described by Ahrens andSeemüller (1992).

The PCR amplification was performed using nested PCR, primed by P1/P7(Schneider et al. 1995, expected size of PCR product – approximately 1800 bp) andfollowed by the phytoplasma universal primer pairs: fA/rA (Ahrens and Seemüller1992, approximately 560 bp) and R16F2n/R16R2 (Lee et al. 1998, approximately1250 bp) or primers specific for the aster yellows group – R16(I)F1/R16(I)16 (Leeet al. 1994, approximately 1100 bp) or apple proliferation group – fAT/rAS (Smartet al. 1996, approximately 500 bp).

Experimental transmission of phytoplasmas... 23

Each PCR mixture (50 l) contained: 1 l of total nucleic acid extraction fromplant tissue, 1.25 l of 10 mM solution of four dNTPs, 0.1 l of 100 M forwardand reverse primers, 1 × DNA polymerase buffer and 1U of HotStarTaq DNA poly-merase (Qiagen, Syngen Biotech, Wrocław, Poland). The following parameterswere used: for direct PCR with primers P1/P7 and nested PCR with primersR16F2n/R16R2 – 35 cycles: 1 min at 94°C, 2 min at 60°C (for P1/P7) and 50°C (forR16F2n/R16R2), 3 min at 72°C; for nested PCR with primers R16(I)F1/R16(I)R1– 35 cycles: 45 s at 94°C, 1 min at 55°C, 2 min at 72°C; for nested PCR with primersfA/rA and fAT/rAS – 25 cycles: 1 min at 95°C, 1 min at 55°C, 1 min at 72°C. Duringthe last cycle, elongation time was extended to 9 min.

PCR products (5 l) were analyzed by 1% agarose gel electrophoresis in 0.5 XTBE buffer followed by staining with ethidium bromide (0.5 g/ml) and visualizedwith UV transilluminator.

RFLP analyses were performed after digestion of 10 l of PCR products (primedby R16F2n/R16R2 primer pair) with single restriction endonucleases AluI, MseI,HpaII (Gibco BRL, Life Technologies, Warsaw, Poland), SspI and HhaI (Roche Di-agnostics, Warsaw, Poland) according to the manufacture’s instructions. The di-gested DNA was resolved by electrophoresis through 6% polyacrylamide gel,stained with ethidium bromide and observed under UV light. The lengths of DNAfragments were estimated by comparison of the position of DNA bands with thoseof molecular weight markers ( X174 DNA/HinfI, Promega Symbios, Gdańsk, Po-land). Phytoplasma 16S rRNA group designations were based on RFLP analysisfollowing the system of Lee et al. (1998).

Results

Phytoplasma detection and identification

The results of our experiments, presented in Tables 1 and 2, demonstrate thatdetection of phytoplasmas using PCR technique in affected magnolias dependsmainly upon time of testing and primer pairs applied. After amplification of DNAisolated from the tested plants with universal primer pair P1/P7, specific DNAband (~1800 bp) was obtained only from samples of reference strains AY1 and AP.No visible product was amplified by direct PCR from samples obtained from mag-nolias and experimentally inoculated or control periwinkle plants.

Nested PCR with universal fA/rA and group 16SrI specific primer pair –R16(I)F1/R16(I)R1 amplified the target sequences from all samples collectedfrom the magnolias in August. In samples collected in October no amplificationwas observed using the primers fA/rA. However, the primer pairsR16(I)F1/R16(I)R1 or F16F2n/R16R2 amplified the target sequences from sam-ples collected in October. After amplification of DNA with group 16SrX specificprimer pair – fAT/rAS, specific DNA bands (~ 500 bp) were obtained. These

24 H. Śliwa and M. Kamińska

bands were observed in samples collected in August from five out of six affectedmagnolia plants but not in samples collected in October (Table 1).

Phytoplasmas were detected in most of the examined samples collected fromexperimentally grafted periwinkles by amplification in nested PCRs with universalfA/rA or R16F2n/R16R2 and groups 16SrX (Phot. 1 a) or 16SrI (Phot. 1 b) specificprimer pairs – R16(I)F1/R16(I)R1 and fAT/rAS (Table 2).

Experimental transmission of phytoplasmas... 25

Table 1Detection of phytoplasmas by the nested PCR in Magnolia liliiflora

cv. ‘Nigra’ source plants

Phytoplasmasource plants

Phytoplasma detection with primer pairs

fA/rA R16(I)F1/R16(I)R1 fA/rA R16F2n/R16R2 R16(I)F1/R16(I)R1 fAT/rAS

12 May 2001 14 August 2001

2

3

8

11

18

20

–

–

–

–

–

–

–

–

–

–

–

–

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

–

+

+

+

+

13 August 2001 16 October 2002

196

214

235

nd

–

+

nd

–

+

–

–

–

+

+

+

+

+

+

–

–

–

nd – not determined.

Table 2Experimental transmission of phytoplasmas from diseased magnolias

to Catharanthus roseus test plants by grafting

Sourceplants

Plants withsymptoms/plants

inoculated

Phytoplasma detection by the nested PCR with primer pairs

fA/rA R16F2n/R16R2 R16(I)F1/R16(I)R1 fAT/rAS

2**

3**

4**

18**

196*

214*

235*

6/8

1/8

3/8

3/8

2/7

4/7

6/8

3/3

0/1

1/3

3/5

3/5

3/5

6/7

3/3

0/1

nd

3/5

3/5

0/2

6/7

3/3

1/1

2/3

1/5

3/5

3/5

6/7

1/3

0/1

1/1

1/3

2/3

0/2

3/5

Total 25/54 19/29 15/23 19/29 8/18

**Grafted on 13 June 2001.**Grafted on 23 August 2001.nd – not determined.

No amplification was observed in nucleic acid extracts in samples collectedfrom the diseased magnolia plants in May and from the asymptomatic magnolias.

Products of PCR primed by R16F2n/R16R2 primer pair from naturally infectedmagnolias and inoculated periwinkles were subjected to RFLP analyses using fiverestriction enzymes (Phot. 2). The restriction patterns obtained after enzymatic di-gestion indicated that the tested plants were infected with two distinct phyto-plasmas: a phytoplasma belonging to subgroup 16SrI-B was detected in allPCR-positive magnolias and experimentally infected periwinkle plants, whereasfive magnolias and eight tested periwinkles were double infected with aster yel-lows and apple proliferation (16SrV-A) phytoplasmas.

26 H. Śliwa and M. Kamińska

Phot. 1. Nested polymerase chain reaction (PCR) amplification of ribosomalDNA fragments obtained with DNA from Catharanthus roseus plants

experimentally infected by grafting (lanes 2–15) using the following primerpair combinations: a) P1/P7 followed by fAT/rAS, b) P1/P7 followed byR16(I)F1/R16(I)R1; M – 1 kb DNA molecular marker (Sigma – Aldrich)

(photo by H. Śliwa)

Phytoplasma transmission

Evaluation of symptoms developed in experimentally grafted periwinkles re-vealed that AY and AP phytoplasmas could be transmitted in summer from all do-nors tested. Three to four months after grafting retarded growth and leaf chlorosiswere observed on single periwinkle plants. Within one year after inoculation, thesesymptoms were followed by leaf chlorosis and diminishing as well as increased pro-duction of the secondary shoots. During winter some of the inoculated plants devel-oped reduced number of small and pale flowers; in summer they did not produceflower symptoms. The most pronounced symptoms developed periwinkles inocu-lated with buds from magnolia No. 235. About 50% of the inoculated periwinkleplants did not develop symptoms within 24 months of observation (Table 2).

Examination of selected recipient periwinkles with PCR showed the presenceof AY or AY and AP phytoplasmas in symptomatic plants, thus confirming the re-sults of visual disease evaluation (Table 2). In a few cases, phytoplasmas were alsodetected in plants, which did not show clear symptoms. None of the uninoculatedperiwinkles developed symptoms.

Discussion

The results obtained in this work indicate that phytoplasmas, responsible formagnolia stunting can be detected in phloem shoots of affected plants using nested

Experimental transmission of phytoplasmas... 27

Phot. 2. Restriction profiles after HhaI, SspI, HpaII, MseI or AluI digestion of the nested PCRproducts (primed by R16F2n/R16R2 primer pair) from magnolia and experimentally infected

Catharanthus roseus; M – molecular weight marker PhiX 174 DNA/HinfI (Promega Symbios), AP– reference strain of apple proliferation phytoplasma, AY1 – reference strain of aster yellows

phytoplasma (photo by H. Śliwa)

PCR. Analyses of samples collected in the middle of August or October showedthat tested magnolias contained mostly phytoplasmas belonging to group 16SrI,subgroup B. Phytoplasmas belonging to the apple proliferation group 16SrX, sub-group A, were found in smaller number of samples collected in August but not inOctober, in plants infected by both phytoplasmas from groups 16SrI and 16SrX.For differentiation of the magnolia phytoplasmas of the AY and AP groups andsubgroups, restriction site analysis using endonucleasis HhaI, SspI proved to behighly suitable (Alma et al. 1996, Marcone et al. 1997 b, Lorenz et al. 1995).

Detectable amount of phytoplasmal DNA was obtained by nested PCR frommajority of magnolia samples collected at the end of summer and in the autumnbut not in the spring. This difficulty in phytoplasma 16SrX detection in late springseems to be common to both pome (Schaper and Seemüller 1984) and stone(Jarausch et al. 1999, Seemüller et al. 1998 b) fruit species. Jarausch et al. (1999)indicated, that in case of ESFY the sampling has to be done between July and Sep-tember. Jarausch et al. (1999) explained these difficulties of phytoplasma detec-tion in Prunus in terms of the limitation of movement from old into new phloemsince the rapid degeneration of old phloem would result in elimination of a consid-erable amount of phytoplasmas.

In our work, efficient detection methods were employed to examine both donormagnolias and recipient periwinkle plants for the presence of phytoplasmas. Theuse of bud wood from carefully selected donor plants appeared to be responsiblefor the high transmission rates achieved in our experiments. Based onsymptomology and PCR-RFLP analyses we stated that AY and AP phytoplasmashave been successfully transmitted by grafting, although the transmission rate ofAY was higher than AP. Studies by several authors have shown that phytoplasmascan be propagated through grafting with phytoplasma infected buds (Seemüller etal. 1998 a, Errea et al. 2002). However, this is the first report of AP and AYphytoplasmas transmitted by grafting to C. roseus test plants. Previously,phytoplasmas belonging to 16SrX were transmitted to C. roseus test plants by psyl-lids (Refatti et al. 1986) and by dodder (Loi et al. 1995, Marcone et al. 1999). Veryrecently, we recorded AY phytoplasma transmission from magnolia to periwinklevia dodder (Kamińska et al. 2001 b) and by grafting from lily and rose plants(Kamińska and Korbin 1999, Kamińska et al. 2001 a).

The experimentally infected periwinkle plants with phytoplasmas from magno-lia showed a reduced vigor, leaf yellowing and diminishing, and flower malforma-tion and discoloration but no virescence or phyllody. Similar symptoms have beenreported to occur in C. roseus plants experimentally infected with AY phytoplasmafrom diseased magnolia (Kamińska et al. 2001 b). The same type of symptomsshowed periwinkles infected with PD and ESFY phytoplasmas from fruit trees(Marcone et al. 1999), with alder yellows phytoplasma from naturally infectedAlnus glutinosa (L.) Gaertn. tree (Marcone et al. 1997 a) or with AY phytoplasmafrom lily (Kamińska and Korbin 1999) and rose plants (Kamińska et al. 2001 a).

Although in the present study a correlation of symptom expression with thepresence of phytoplasma in experimentally infected C. roseus plants is difficult toestablish, the results confirm these from previous study of dodder inoculation and

28 H. Śliwa and M. Kamińska

strongly suggest that typical symptoms like leaf chlorosis and diminishing andshoot proliferation are highly correlated to the presence of AY and APphytoplasmas.

The results obtained in this study provide good basis for the development ofsampling protocols for routine diagnosis of AY and AP phytoplasmas in Magnoliaspecies by PCR. Indications from these and other unpublished results of the au-thors suggest that in order to obtain reliable results sampling has to be done in Au-gust–October. The insect vectors of AY and AP phytoplasmas in Magnolia spp.plants are unknown. It seems likely that the efficient acquisition occurs at the endof summer when the phytoplasma titres are high.

Summary

Phytoplasmas detected in magnolia plants with stunting symptoms were classi-fied as aster yellows phytoplasma (16SrI-B) and apple proliferation phytoplasma(16SrX-A) on the basis of restriction fragment length polymorphism (RFLP) anal-ysis of PCR-amplified 16S rDNA phytoplasma fragments. The phytoplasmas weresuccessfully transmitted to Catharanthus roseus test plants by grafting, and the infec-tion was confirmed by symptom expression and phytoplasma specific PCR andRFLP analysis.

Streszczenie

EKSPERYMENTALNE PRZENIESIENIE FITOPLAZM Z CHORYCHMAGNOLII NA ROŚLINĘ TESTOWĄ CATHARANTHUS ROSEUS

PRZEZ SZCZEPIENIE

Fitoplazmy występujące w roślinach magnolii z objawami karłowatości zostałyzaklasyfikowane jako fitoplazma żółtaczki astra (16SrI-B) i fitoplazma proliferacjijabłoni (16SrX-A). Identyfikacji dokonano za pomocą analizy polimorfizmu długo-ści fragmentów restrykcyjnych (RFLP) produktów łańcuchowej reakcji polimerazy(PCR) ze starterami umożliwiającymi amplifikację fragmentu genu 16S rDNA fito-plazm. Fitoplazmy przeniesiono z chorych magnolii na siewki rośliny testowej ka-taranta różowego (Catharanthus roseus) techniką szczepienia. Porażenie roślinbarwinka przez fitoplazmy zostało potwierdzone występowaniem charakterystycz-nych objawów chorobowych oraz wynikami analizy PCR-RFLP.

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Authors’ address: Dr Hanna Śliwa,Prof. dr hab. Maria Kamińska,Research Institute of Pomology and Floriculture,ul. Pomologiczna 18,96-100 Skierniewice,Polande-mail: hsliwa@insad.pl

Accepted for publication: 25.03.2004

Experimental transmission of phytoplasmas... 31