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Blackwell Publishing, Ltd.Oxford, UKEPPBulletin OEPP/EPPO Bulletin0250-8052OEPP/EPPO, 2005Original ArticleDiagnosticsDiagnostics

Organisation Européenne et Méditerranéenne pour la Protection des PlantesEuropean and Mediterranean Plant Protection Organization

Normes OEPP EPPO Standards

DiagnosticsDiagnostic

PM 7/47

Organisation Européenne et Méditerranéenne pour la Protection des Plantes1, rue Le Nôtre, 75016 Paris, France

272 Diagnostics

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Approval

EPPO Standards are approved by EPPO Council. The date ofapproval appears in each individual standard. In the terms ofArticle II of the IPPC, EPPO Standards are Regional Standardsfor the members of EPPO.

Review

EPPO Standards are subject to periodic review and amend-ment. The next review date for this EPPO Standard isdecided by the EPPO Working Party on PhytosanitaryRegulations.

Amendment record

Amendments will be issued as necessary, numbered and dated.The dates of amendment appear in each individual standard (asappropriate).

Distribution

EPPO Standards are distributed by the EPPO Secretariat toall EPPO member governments. Copies are available to anyinterested person under particular conditions upon request tothe EPPO Secretariat.

Scope

EPPO Standards on Diagnostics are intended to be used byNPPOs in their capacity as bodies responsible for theapplication of phytosanitary measures. Standards on diagnosticprotocols are concerned with the diagnosis of individual pestsand describe different methods which can be used to detect andidentify pests of phytosanitary concern for the EPPO region.General Standards on diagnostics are in preparation on: (1) thepurpose of diagnostic protocols (which may differ according tothe circumstances of their use); and (2) reporting and docu-mentation of diagnoses.

In 1998, EPPO started a new programme to prepare diagnosticprotocols for the regulated pests of the EPPO region (includingthe EU). The work is conducted by the EPPO Panel on Diag-nostics and other specialist Panels. The objective of the pro-gramme is to develop an internationally agreed diagnosticprotocol for each regulated pest. The protocols are based on themany years of experience of EPPO experts. The first drafts areprepared by an assigned expert author(s). They are writtenaccording to a ‘common format and content of a diagnosticprotocol’ agreed by the Panel on Diagnostics, modified asnecessary to fit individual pests. As a general rule, the protocolrecommends a particular means of detection or identificationwhich is considered to have advantages (of reliability, easeof use etc.) over other methods. Other methods may alsobe mentioned, giving their advantages/disadvantages. If amethod not mentioned in the protocol is used, it should bejustified.

The following general provisions apply to all EPPOStandards on Diagnostics:• laboratory tests may involve the use of chemicals or appara-

tus which present a certain hazard. In all cases, local safetyprocedures should be strictly followed

• use of names of chemicals or equipment in these EPPOStandards implies no approval of them to the exclusion ofothers that may also be suitable

• laboratory procedures presented in the protocols may beadjusted to the standards of individual laboratories, providedthat they are adequately validated or that proper positive andnegative controls are included.

References

EPPO/CABI (1996)

Quarantine Pests for Europe

, 2nd edn. CAB Interna-tional, Wallingford (GB).

EU (2000) Council Directive 2000/29/EC of 8 May 2000 on protectivemeasures against the introduction into the Community of organismsharmful to plants or plant products and against their spread within theCommunity.

Official Journal of the European Communities

L169, 1–112.

FAO (1997)

International Plant Protection Convention

(new revised text).FAO, Rome (IT).

IPPC (1993)

Principles of plant quarantine as related to international trade

.ISPM no. 1. IPPC Secretariat, FAO, Rome (IT).

IPPC (2002)

Glossary of phytosanitary terms

. ISPM no. 5. IPPC Secretariat,FAO, Rome (IT).

OEPP/EPPO (2003) EPPO Standards PM 1/2(12): EPPO A1 and A2 lists ofquarantine pests.

EPPO Standards PM1 General phytosanitarymeasures

, 5–17. OEPP/EPPO, Paris (FR).

Definitions

Regulated pest

: a quarantine pest or regulated non-quarantine pest.

Quarantine pest

: a pest of potential economic importance to thearea endangered thereby and not yet present there, or presentbut not widely distributed and being officially controlled.

Outline of requirements

EPPO Standards on Diagnostics provide all the informationnecessary for a named pest to be detected and positivelyidentified by an expert (i.e. a specialist in entomologist,mycology, virology, bacteriology, etc.). Each protocol beginswith some short general information on the pest (itsappearance, relationship with other organisms, host range,effects on host, geographical distribution and its identity) andthen gives details on the detection, identification, comparisonwith similar species, requirements for a positive diagnosis,list of institutes or individuals where further information onthat organism can be obtained, references (on the diagnosis,detection/extraction method, test methods).

Existing EPPO Standards in this series

Forty-one EPPO standards on diagnostic protocols havealready been approved and published. Each standard is

Diagnostics 273

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numbered in the style PM 7/4 (1), meaning an EPPO Standardon Phytosanitary Measures (PM), in series no. 7 (DiagnosticProtocols), in this case standard no. 4, first version. The existingstandards are:PM 7/1 (1)

Ceratocystis fagacearum

.

Bulletin OEPP/EPPOBulletin

31

, 41–44PM 7/2 (1)

Tobacco ringspot nepovirus

.

Bulletin OEPP/EPPOBulletin

31

, 45–51PM 7/3 (1)

Thrips palmi

.

Bulletin OEPP/EPPO Bulletin

31

, 53–60PM 7/4 (1)

Bursaphelenchus xylophilus. Bulletin OEPP/EPPOBulletin

31

, 61–69PM 7/5 (1)

Nacobbus aberrans. Bulletin OEPP/EPPO Bulletin

31

, 71–77PM 7/6 (1)

Chrysanthemum stunt pospiviroid. Bulletin OEPP/EPPO Bulletin

32

, 245–253PM 7/7 (1)

Aleurocanthus spiniferus. Bulletin OEPP/EPPOBulletin

32

, 255–259PM 7

/

8 (1)

Aleurocanthus woglumi. Bulletin OEPP/EPPOBulletin

32

, 261–265PM 7/9 (1)

Cacoecimorpha pronubana. Bulletin OEPP/EPPOBulletin

32

, 267–275PM 7/10 (1)

Cacyreus marshalli. Bulletin OEPP/EPPO Bulletin

32

, 277–279PM 7/11 (1)

Frankliniella occidentalis. Bulletin OEPP/EPPOBulletin

32

, 281–292PM 7/12 (1)

Parasaissetia nigra. Bulletin OEPP/EPPO Bulletin

32

, 293–298PM 7/13 (1)

Trogoderma granarium. Bulletin OEPP/EPPOBulletin

32

, 299–310PM 7/14 (1)

Ceratocystis fimbriata

f. sp.

platani. BulletinOEPP/EPPO Bulletin

33

, 249–256PM 7/15 (1)

Ciborinia camelliae. Bulletin OEPP/EPPO Bulletin

33

, 257–264PM 7/16 (1)

Fusarium oxysporum

f. sp.

albedinis. BulletinOEPP/EPPO Bulletin

33

, 265–270PM 7/17 (1)

Guignardia citricarpa. Bulletin OEPP/EPPOBulletin

33

, 271–280PM 7/18 (1)

Monilinia fructicola. Bulletin OEPP/EPPOBulletin

33

, 281–288PM 7/19 (1)

Helicoverpa armigera. Bulletin OEPP/EPPOBulletin

33

, 289–296PM 7/20 (1)

Erwinia amylovora. Bulletin OEPP/EPPO Bulletin

34

, 159–172PM 7/21 (1)

Ralstonia solanacearum. Bulletin OEPP/EPPOBulletin

34

, 173–178PM 7/22 (1)

Xanthomonas arboricola

pv.

corylina. BulletinOEPP/EPPO Bulletin

34

, 179–182PM 7/23 (1)

Xanthomonas axonopodis

pv.

dieffenbachiae.Bulletin OEPP/EPPO Bulletin

34

, 183–186PM 7/24 (1)

Xylella fastidiosa. Bulletin OEPP/EPPO Bulletin

34

, 187–192

PM 7/25 (1)

Glomerella acutata. Bulletin OEPP/EPPO Bulletin

34

, 193–200PM 7/26 (1)

Phytophthora cinnamomi. Bulletin OEPP/EPPOBulletin

34

, 201–208PM 7/27 (1)

Puccinia horiana. Bulletin OEPP/EPPO Bulletin

34

, 209–212PM 7/28 (1)

Synchytrium endobioticum. Bulletin OEPP/EPPOBulletin

34

, 213–218PM 7/29 (1)

Tilletia indica. Bulletin OEPP/EPPO Bulletin

34

,219–228

PM 7/30 (1)

Beet necrotic yellow vein benyvirus. BulletinOEPP/EPPO Bulletin

34

, 229–238PM 7/31 (1)

Citrus tristeza closterovirus. Bulletin OEPP/EPPO Bulletin

34

, 239–246PM 7/32 (1)

Plum pox potyvirus. Bulletin OEPP/EPPO Bulletin

34

, 247–256PM 7/33 (1)

Potato spindle tuber pospiviroid. Bulletin OEPP/EPPO Bulletin

34

, 257–270PM 7/34 (1)

Tomato spotted wilt tospovirus. Bulletin OEPP/EPPO Bulletin

34

, 271–280PM 7/35 (1)

Bemisia tabaci. Bulletin OEPP/EPPO Bulletin

34

,281–288

PM 7/36 (1)

Diabrotica virgifera. Bulletin OEPP/EPPOBulletin

34

, 289–294PM 7/37 (1)

Thaumetopoea pityocampa. Bulletin OEPP/EPPO Bulletin

34

, 295–298PM 7/38 (1)

Unaspis citri. Bulletin OEPP/EPPO Bulletin

34

,299–302

PM 7/39 (1)

Aphelenchoides besseyi. Bulletin OEPP/EPPOBulletin

34

, 303–308PM 7/40 (1)

Globodera rostochiensis

and

Globodera pallida.Bulletin OEPP/EPPO Bulletin

34

, 309–314PM 7/41 (1)

Meloidogyne chitwoodi

and

Meloidogyne fallax.Bulletin OEPP/EPPO Bulletin

34

, 315–320Some of the Standards of the present set result from adifferent drafting and consultation procedure. They are theoutput of the DIAGPRO Project of the Commission of theEuropean Union (no. SMT 4-CT98-2252). This projectinvolved four ‘contractor’ diagnostic laboratories (in England,Netherlands, Scotland, Spain) and 50 ‘inter-comparison’laboratories in many European countries (within and outsidethe European Union), which were involved in ring-testing thedraft protocols. The DIAGPRO project was set up in fullknowledge of the parallel activity of the EPPO WorkingParty on Phytosanitary Regulations in drafting diagnosticprotocols, and covered regulated pests which were for thatreason not included in the EPPO programme. The DIAGPROprotocols have been approved by the Council of EPPO asEPPO Standards in series PM 7. They will in future besubject to review by EPPO procedures, on the same termsas other members of the series.

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303

Blackwell Publishing, Ltd.Oxford, UKEPPBulletin OEPP/EPPO Bulletin0250-8052OEPP/EPPO, 200535Original ArticleDiagnostics

Mycosphaerella pini

European and Mediterranean Plant Protection Organization PM 7/47(1)Organisation Européenne et Méditerranéenne pour la Protection des Plantes

Diagnostics

1

Diagnostic

Mycosphaerella pini

Specific scope

This standard describes a diagnostic protocol for

Mycosphaerellapini

.

Specific approval and amendment

Approved in 2004-09.

Introduction

Mycosphaerella pini

is the cause of red band needle blight ofpine and retards growth of many pine species. The fungus killsfoliage and attacks over several years can kill the trees.

M. pini

mainly attacks

Pinus

spp. In rare cases,

Pseudotsuga menziesii

,

Larix decidua

and

Picea abies

growing right next to severelydiseased pines have been reported to be infected.

M. pini

hasbecome the most damaging pathogen on pines in the SouthernHemisphere, and is also spreading in Europe on several pinespecies. The fungus is usually encountered in its conidial state,

Dothistroma septospora

.

Identity

Name:

Mycosphaerella pini

E. Rostrup apud Munk

Synonym:

Scirrhia pini

Funk & A. K. Parker

Anamorph:

Dothistroma septospora

(G. Doroguine) Morelet

Synonyms:

Dothistroma pini

Hulbary,

Cytosporina septospora

G. Doroguine

Taxonomic position:

Fungi

:

Ascomycetes

:

Dothideales

EPPO computer code:

SCIRPI

Phytosanitary categorization:

EU Annex designation: II/A2 –as

Scirrhia pini

Detection

Symptoms

Disease symptoms first appear on pine needles as yellow spots(Web Fig. 1a–d) that spread to become a necrotic band. Thetransition from lesions to healthy green tissue is abrupt. By

further development, the necroses enlarge to bands that encirclethe needle and cause death of parts beyond the bands. Diseasedneedles often show dead tips, central zones with lesions ornecroses, and green bases (Web Fig. 2). The brown, dead partsof the needle tissue typically develop a conspicuous reddening,in the form of bands around the needle axis (Web Fig. 3a,b).This red colour is due to accumulation of dothistromin, a toxinthe fungus produces in needle tissue and culture. However, thisfeature is not always present on every pine species and location.Strong light enhances reddish symptoms caused by the toxin,and shade suppresses them. In some cases the reddening is reducedto small parts surrounding the fructifications (Web Fig. 4a,b;hand lens 10

×

). If the characteristic red bands are not produced,or are suppressed, so that no reddening is visible, the macroscopicsymptoms at the beginning of disease development can easilybe confused with those due to

Mycosphaerella dearnessii

(brownspot needle blight) or

Mycosphaerella gibsonii

. In the brown andred coloured dead parts of the needle, the black stroma of thefructification develops under the epidermis, visible as round blacksubepidermal spots (Web Fig. 5). During further development,the elliptical fruit bodies, arranged in parallel to the long axis of theneedle, break through the epidermis opening by a longitudinalslit, or two slits, raising a flap of epidermal and hypodermaltissue. Sometimes, the conidiomata are found concentrated inthe red bands. After severe attack, whole needles turn brownand drop prematurely, the older ones first. In less severe attacks,needle fall may be delayed for one or two years. Pines withsevere attack typically show twigs with only last year’s needles,looking like a paintbrush (Web Fig. 6). Over several years thismay result in branch and tree death. See Sinclair

et al.

(1989).

Isolation

The fungus is most easily identified by examination ofmature conidiomata (and can also be isolated directly from

1

The Figures in this Standard marked ‘Web Fig.’ are published on the EPPOwebsite www.eppo.org.

304

Mycosphaerella pini

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them). If none are present, and there are no characteristic redbands visible, the fungus should be isolated from the needles,which is possible, but difficult. Samples are best taken fromaffected needles with dead tips and necroses or lesions in thecentral zone of the needle. After surface sterilization, needlesare cut into segments 4–6 mm long under sterile conditions,placed on malt extract agar medium (MEA: 2% malt extract, 2%agar agar) or MEA + chloramphenicol (1 ppm) in 9-cm Petridishes. After an incubation period of 2–4 weeks roomtemperature (20

°

C) in daylight, isolates can be examined.Mycelium usually appears on the two cut surfaces of the needlesegment. If both fast and slow-growing fungi are visible, theslow-growing mycelia should be subcultured onto new MEAPetri dishes. If fruit bodies and conidia do not form in culture,there are two methods for stimulating production ofconidiomata. Autoclaved pine needles can be added next to thesubcultured fungus. In addition, black light (near UV, emissionmaximum about 360 nm) can enhance the formation of fruitbodies and sporulation after continuous storage at 15

°

C. Oftenincubation in the cold (refrigerator, 4–6

°

C) for 10–14 daysfollowed by 10–14 days in the warm (20

°

C) provides goodconditions for production of conidiomata. Different strains of

M. pini

behave differently, so no one of these methods can befavoured over the other. In some cases, methods can becombined.

Because of slow growth in culture and the presence of endo-phytic fungi in pine needles, isolation is not always successful.Other fungi or bacteria present in the needles may grow rapidlyon the culture medium and mask any possible colonies of thepathogen.

Method for surface sterilization of pine needles

Needles are immersed in 70% ethanol for 30 s, or 2% NaOClfor 60 s, or 96% ethanol for 10 s, then rinsed in sterile water.

Identification

Identification on the basis of morphological features

A quick presumptive indication of the presence of

Mycosphaerella pini

in pine needles is the existence of areddish tint in the necrotic needle tissue, often visible as redcoloured bands around the needle axis (Web Fig. 3a,b). Finalconfirmation can only be obtained by microscopic identificationof the conidial stage when the typical conidia are produced.Ascostromata (Web Figs 9 and 10) are produced irregularly onfallen needles and are not particularly useful for identification,unless red bands are present at the same time.

Mature conidiomata split the epidermis irregularly or by onelateral or median longitudinal slit, seldom by two slits and pro-duce a slimy whitish-hyaline spore mass under moist condi-tions (Web Fig. 7, hand lens 10

×

). On samples, sporulation canbe induced by placing needles with mature conidiomata in amoist chamber for one or two days. The conidia are hyaline,thin-walled, smooth, straight to curved, fusiform to slightly cla-vate, 1–5 septate, 12–28–48

×

2–3

µ

m with both rounded apexand sometimes truncate base (Web Fig. 8).

If no mature conidiomata are present, isolation of the fun-gus is possible (see Detection).

M. pini

can then be identifiedfrom conidia produced in culture. On MEA, first a white aerial

Table 1 Morphology and diagnostic features of Mycosphaerella pini

TeleomorphOccurrence Irregularly dispersed on dead needle tissue, typically aggregated in red bands, most on fallen needles, seldom developed

Ascostromata Subepidermal development, becoming erumpent and splitting the epidermis irregularly or by one or two longitudinal slits when mature, composed of pseudoparenchymatic, thick-walled cells, uni- to multiloculate, black, 400–1000 × 300–400 µm (Web Fig. 10)

Fructification Pseudothecium (ascolocular development)

Loculi Globose to pear-shaped, ostiolate, periphysate, 70–110 × 70–90 µm

Asci Saccate to cylindrical, bitunicate, 8-spored, hyaline, with a rounded apex, 35–55 × 6–9 µm (Web Fig. 9)

Ascospores Elliptic, 1-septate, hyaline, typically 4-guttulate, 10–15 × 3–4 µm (Web Fig. 9)

Spermatia Rod-shaped, hyaline, 1.5–2.5 × 0.5–1 µm

AnamorphOccurence Irregularly dispersed on necrotic areas, red bands and parts of dead needle tissue, sometimes aggregated in red bands

Conidiomata Subepidermal development, becoming erumpent and rupturing the epidermis irregularly or by one lateral or median longitudinal slit, seldom by two slits, when mature, elliptical, arranged parallel to the long axis of the needle, black, basal stroma composed of thick-walled pseudoparenchymatic cells, 300–650 × 150–300 µm (Web Figs 4a,b and 11)

Fructification Variable, from pseudopycnidial to acervular

Conidia Exuded in a whitish mucilaginous mass; fusiform to short-clavate, hyaline, smooth, thin-walled, 1–5 septate, most 2–3 septate, rounded apex and truncate base, 12–28–48 × 2–3 µm (Web Fig. 8)

Culture On malt extract agar (2% malt extract, 2% agar) 1.5–2 mm growth/week at 20°C, colonies stromatic, brown to grey-black, producing whitish conidial slime, agar coloured light reddish-brown by diffusates

Diagnostics 305

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mycelium appears which later turns grey-brown, forming stro-matic and erumpent colonies. At 20

°

C, the mycelium grows1.5–2 mm a week. Conidia of the anamorph (

Dothistromaseptospora

) are produced, visible as whitish-hyaline slimyspore masses. Colonies produce light reddish-brown diffu-sates on MEA and sometimes red pigments are visible. Fullmorphological and diagnostic features are listed in Table 1. SeeButin (1984).

Comparison with similar species

Besides

M. pini

, two other

Mycosphaerella

spp. have beendescribed on pines:

M. dearnessii

(anamorph:

Lecanostictaacicola

) and

M. gibsonii

(anamorph:

Pseudocercospora pini-densiflorae

) (Evans, 1984; Pehl & Wulf, 2001). Most of thestages of disease development are very similar in symptoms andmorphological features for the three fungi. Their teleomorphs(if available for examination) are very similar morphologically,and can hardly be distinguished in the absence of some othercharacteristic information, such as the profuse reddish tint of thenecrotic needle tissue characteristic by attack by

M. pini

.However, if there are clear red bands visible, together withascomata or conidiomata,

M. pini

can be identified withoutdoubt, since

M. dearnessii

and

M. gibsonii

never produce aclear red colouring on attacked pine needles.

The anamorphs are consistently different and provide thebest characters to separate the species. The conidiomata knownas

Lecanosticta

and

Dothistroma

vary in form between acervuliand pseudopycnidia, according to host and climate, but differclearly from the fruit bodies of

Pseudocercospora

, which aresporodochia. The most important and consistent character todistinguish

M. pini

from

M. dearnessii

is the nature of the sporewall of the conidium. The conidia of

M. pini

(of

Dothistroma

type), viewed under the microscope, are hyaline, thin-walledand smooth, whereas those of

M. dearnessii

(of

Lecanosticta

type) have melanin granules integrated in their outer wall, sothat the spores are pigmented, thick-walled with a verrucosesurface. The best view is obtained using differential inter-ference contrast optics at an magnification over

×

400. Theconidia of

M. pini

do not, however, differ distinctly in size fromthose of

M. dearnessii

.The three fungi can also be distinguished by cultural

characteristics. On MEA, colonies of

M. pini

are slow-growing,grey-brown-black, stromatic and produce a whitish conidial slime.A reddish-brown diffusate is present in the agar.

M. dearnessii

grows significantly larger on the same media and showsgreen-black stromatic colonies with an olive green conidial slime.Typically a yellow diffusate is visible.

M. gibsonii

is the fastestgrowing species and forms grey-coloured colonies with a woollyaerial mycelium.

Identification by molecular biological methods

Pehl

et al

. (2004) have used a PCR-based ITS-RFLP techniquefor differentiating

M. pini

from

M. dearnessii

and 10 otherfungi frequently occurring in Europe on pine needles. For

details of this technique, see EPPO Standard PM 7/46Diagnostic protocol for

Mycosphaerella dearnessii

(OEPP/EPPO, 2005).

Conclusion

There are three morphological methods available (

in situ

morphology of fungal organs present on the sample as it isreceived in the laboratory; moist chamber incubation followedby examination for fungal organs produced during incubationof the sample, isolation of the fungus followed by examinationof fungal organs produced on agar media) and one molecularmethod (PCR-based ITS-RFLP). All are equivalent and eachmethod is adequate by itself. At least one of these methodsshould have been positive for a positive diagnosis. The essentialdistinguishing features of

M. pini

are the conidia of itsanamorph

D. septospora

produced on mature conidiomata oninfected needles and in culture. Because of the limitations ofclassical identification methods, the use of PCR-based ITS-RFLP allows fast and reliable identification of

M. pini

.Symptoms on pines infected by

M. pini

and adequate iden-tification methods to be undertaken are shown in Web Fig. 13.

Reference culture

D 291.

Reporting and documentation

Guidance on reporting and documentation is given in EPPOStandard PM7/– (in preparation).

Further information

Further information on this organism can be obtained from:Dr L. Pehl, Federal Biological Research Centre for

Agriculture and Forestry, Institute for Plant Protection inForests, Messeweg 11/12, D-38104 Braunschweig (Germany)

Dr T. L. Cech, Bundesamt und Forschungszentrum für Wald,Institut für Forstschutz, Abteilung für Phytopathologie,Seckendorff-Gudent-Weg 8, A-1131 Wien (Austria)

Prof Dr Ottmar Holdenrieder, ETH-Zentrum, DepartmentWald- und Holzforschung, Rämistr. 101, 8092 Zürich(Switzerland)

Prof Dr T. Kowalski, Akademia Rolnicza, im. H. Ko

44a

taja,Wydzia

4

Le

s

ny, Katedra Fitopatologii Le

s

nej, Al. 29Listopada 46, 31–425 Kraków (Poland).

Acknowledgements

This protocol was originally drafted by Dr L. Pehl, BBA,Braunschweig (DE).

References

Butin H (1984) [Teleomorph and anamorph development in

Scirrhia pini

onneedles on

Pinus nigra

.]

Sydowia

38

, 20–27.

Diagnostics 306

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, 303–306

Evans HC (1984) The genus

Mycosphaerella and its anamorphs Cercoseptoria,Dothistroma and Lecanosticta on pines. Mycological Papers 153, 1–82.

OEPP/EPPO (2005) EPPO Standards PM 7/46 Diagnostics Mycosphaerelladearnessii. Bulletin OEPP/EPPO Bulletin 35, 299–302.

Pehl L, Burgermeister W & Wulf A (2004) [Mycosphaerella needle blightsof pine – identification by ITS-RFLP patterns.] Nachrichtenblatt DesDeutschen Pflanzenschutzdienstes 56, 239–244 (in German).

Pehl L & Wulf A (2001) [Mycosphaerella needle blights of pine – symptoms,biology and differential diagnosis.] Nachrichtenblatt Des DeutschenPflanzenschutzdienstes 53, 217–222 (in German).

Sinclair WA, Lyon H & Johnson WT (1989) Diseases of Trees and Shrubs,2nd edn, pp. 48–49. Cornell University Press, New York (US).

Web Figs. 1a-d: Yellow spots and necrotic bands on needles of Pinus cembra (1a, d) and P. mugo (1b, c) caused by Mycosphaerella pini (Dothistroma septospora)

Web Fig. 2: Needles of Pinus mugo infected by Mycosphaerella pini (Dothistromaseptospora).

Web Fig. 3a, b: Typical red bands on needles of Pinus nigra caused by Mycospaerella pini (Dothistroma septospora).

Web Fig. 4a, b: Fructifications of Mycosphaerella pini rupturing needle epidermis of Pinus mugo. Red pigments are visible (lens).

Web Fig. 5: Spots of black stroma of Mycosphaerella pini (Dothistromaseptospora) developing under the needle epidermis (Pinus mugo).

Web Fig. 6: Attack of Mycosphaerella pini (Dothistroma septospora) on Pinusmugo over several years showing only last years needles (paintbrush-looking).

Web Fig. 7: Slimy-hyaline spore mass of Dothistroma septospora producedfrom conidiomata under moist conditions.

Web Fig. 8: Conidia of Dothistroma septospora.

Web Fig. 9: Asci and ascospores of Mycosphaerella pini (staining: cotton blue in lactic acid).

Web Fig 10: Cross section through an ascoma of Mycosphaerella pini(staining: thionine).

Web Fig. 11: Cross section through a conidioma of Dothistromaseptospora (staining: thionine)

600 bp

M 1 2 3 4 5 6 M M 1 2 3 4 5 6 M

Dothistroma septospora Lecanosticta acicola

Web Fig. 12: ITS-RFLP patterns of a Dothistroma septospora isolatecompared with a Lecanosticta acicola isolate. M) 100 bp marker, 1) ITS-Amplicon, 2) Hinf I, 3) Hae III, 4) Hha I, 5) Nci I, 6) Hpa II.

Web Fig. 13 Symptoms on pines infected by M. pini (Dothistroma septospora)

Suspicious symptoms Identification scheme

Pine trees with strong needle cast. Twigs looking often like paint-brushes.

Needles with necroses and red bands (strong indication for M. pini), stroma or fruit bodies especially in the litter. Further studies (microscope or PCR / RFLP) necessary.

Needles with red bands (strong indication for M. pini), stroma or fruit bodies. Further research (microscopically studies or PCR / RFLP) necessary.

Twigs with needles showing dead tips or greater necrotic parts, often with green needle basis.

Needles with yellow spots or bands especially on current-years-old needles in spring and mid summer.

Further research is necessary by isolation of the pathogen on culture media. Comparison with reference culture or PCR / RFLP.

Needles with brown necrotic tissue, showing black spots of stroma developing under the epidermis.

Further research is necessary by isolation of the pathogen on culture media or direct evidence by PCR / RFLP procedure.

Needles with brown necrotic tissue, showing fruit bodies breaking through the epidermis by longitudinal slits, raising a flap of epidermis.

Strong indication for M. pini if red pigments on the fruit bodies are visible (lens 10x). Microsco-pically examination for conidia or ascospores is necessary. Moist chamber, if no spores are present, or direct evidence by PCR / RFLP procedure.

Strong indication for M. pini. Further research for fruit bodies and spores is necessary.

Needles with brown necrotic tissue, showing typical red bands.