Septoria-like pathogens causing leaf and fruit spot of pistachio.

Several species of Septoria are associated with leaf and fruit spot of pistachio (Pistacia vera), though their identity has always been confused, making identification problematic. The present study elucidates the taxonomy of the Septoria spp. associated with pistachio, and distinguishes four species associated with this host genus. Partial nucleotide sequence data for five gene loci, ITS, LSU, EF-1α, RPB2 and Btub were generated for a subset of isolates. Cylindroseptoria pistaciae, which is associated with leaf spots of Pistacia lentiscus in Spain, is characterised by pycnidial conidiomata that give rise to cylindrical, aseptate conidia. Two species of Septoria s. str. are also recognised on pistachio, S. pistaciarum, and S. pistaciae. The latter is part of the S. protearum species complex, and appears to be a wide host range pathogen occurring on hosts in several different plant families. Septoria pistacina, a major pathogen of pistachio in Turkey, is shown to belong to Pseudocercospora, and not Septoria as earlier suspected. Other than for its pycnidial conidiomata, it is a typical species of Pseudocercospora based on its smooth, pigmented conidiogenous cells and septate conidia. This phenomenon has also been observed in Pallidocercospora, and seriously questions the value of conidiomatal structure at generic level, which has traditionally been used to separate hyphomycetous from coelomycetous ascomycetes. Other than DNA barcodes to facilitate the molecular identification of these taxa occurring on pistachio, a key is also provided to distinguish species based on morphology.

INTRODUCTION

The genus Pistacia (Anacardiaceae), which is believed to have originated in Central Asia, consists of at least 11 species (Parfitt & Badenes 1997). Among these, Pistacia vera (pistachio), which is native to Western Asia and parts of the Middle East, is the only cultivated and economically important species (Tous & Ferguson 1996). Several important plant pathogens have been recorded from pistachio, causing fruit and root rot, blossom and shoot blight, canker and rust, and other problems (Michailides , http://nt.ars-grin.gov/fungaldatabases/). Of these, Septoria leaf spot is one of the more important diseases associated with fruit and leaf spot.

Desmaziéres (1842) published the first description of a Septoria species causing a leaf spot of Pistacia vera in northern France, for which he introduced the name S. pistaciae. In the same year, Léveillé (1842) described and illustrated Dothidea pistaciae causing a leaf spot of a Pistacia sp. in Crimea. Cooke (1884), upon examination of the type material on which Léveillé based D. pistaciae, transferred it to Septoria. Apparently Cooke was unaware of the existence of Desmaziéres’ name. Allescher (1901) proposed the binominal S. pistacina to replace S. pistaciae (Lév.) Cooke 1884 and to differentiate it from S. pistaciae Desm. 1842. Caracciolo (1934) reported a third species from pistachio as causing a serious leaf spot in Sicily, which he subsequently described as S. pistaciarum. Finally, Chitzanidis (1956) reported sexual morphs for two of the three species, namely Mycosphaerella pistacina (for Septoria pistacina) and Mycosphaerella pistaciarum (for Septoria pistaciarum) (Teviotdale ).

Septoria pistaciarum is known from the USA, and the eastern Mediterranean and southeast Anatolian regions (Young & Michailides 1989). Septoria pistaciae is known from the USA (Califonia), Asia (Armenia, Republic of Georgia, India, Israel, Kazakhstan, Kirgizstan, Syria, Tadjikistan, Turkey, Turkmenistan and Uzbekistan), Europe (Albania, France, Greece, Italy, Portugal and Ukraine), and Africa (Egypt) (Pantidou 1973, Dudka , Andrianova & Minter 2004, Haggag ). Septoria pistacina is known from Greece (Chitzanidis 1956), Syria, Turkey and Iran (Aghajani ), and appears to have a more limited distribution.

The application of these Septoria names to the respective diseases that they are associated with has been plagued by confusion ever since they were first introduced. The aim of the present study is to elucidate the taxonomy of the Septoria species associated with fruit and leaf spot diseases of pistachio, and to place them in a phylogenetic context within Mycosphaerellaceae.

MATERIALS AND METHODS

Isolates

Isolations were made from leaf spots by placing leaves in damp chambers for 1–2 d to enhance sporulation. Single conidial colonies were established from sporulating conidiomata on Petri dishes containing 2 % malt extract agar (MEA) as described earlier (Crous ). Additional strains were obtained from the culture collection of the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands. Colonies were subcultured onto potato-dextrose agar (PDA), oatmeal agar (OA), and MEA (Crous ), and incubated at 25 °C under continuous near-ultraviolet light to promote sporulation. Reference strains were deposited at the CBS (Table 1).

Table 1.

Collection details and GenBank accession numbers of isolates included in this study.

Species	Isolate no.1	Host	Location	Collected by	GenBank accession no.2
					RPB2	LSU	ITS	Btub	EF-1α
Caryophylloseptoria lychnidis	CBS 109098	Silene pratensis	Austria	G. Verkley	KF252292	KF251790	–	–	–
Caryophylloseptoria silenes	CBS 109100	Silene nutans	Austria	G. Verkley	KF252298	KF251796	–	–	–
	CBS 109103	Silene pratensis	Austria	G. Verkley	KF252299	KF251797	–	–	–
Caryophylloseptoria spergulae	CBS 109010	Spergula morisonii	Netherlands	A. Aptroot	KF252487	KF251995	–	–	–
	CBS 397.52	Dianthus caryophyllus	Netherlands	Schouten	KF252301	KF251799	–	–	–
Cercospora apii	CBS 118712	–	Fiji	P. Tyler	KF252302	KF251800	–	–	–
Cercospora ariminensis	CBS 137.56	Hedysarum coronarium	Italy	M. Ribaldi	KF252303	KF251801	–	–	–
Cercospora beticola	CBS 124.31	Beta vulgaris	Romania	–	KF252155	KF251650	–	–	–
Cercospora zebrina	CBS 118790	Trifolium subterraneum	Australia	M.J. Barbetti	KF252156	KF251651	–	–	–
Cylindroseptoria pistaciae	CBS 471.69	Pistacia lentiscus	Spain	H.A. van der Aa	KF252161	KF251656	–	–	–
Pallidocercospora acaciigena	CBS 112515	Acacia mangium	Venezuela	M.J. Wingfield	KF442687	KF442656	–	–	–
	CBS 112516	Acacia mangium	Venezuela	M.J. Wingfield	KF442688	GU253697	–	–	–
Pallidocercospora colombiensis	CBS 110968	Eucalyptus urophylla	Colombia	M.J. Wingfield	KF442689	AY752148	–	–	–
	CBS 110969	Eucalyptus urophylla	Colombia	M.J. Wingfield	KF442690	KF442657	–	–	–
Pallidocercospora crystallina	CBS 110699	Leucospermum	USA	P.W. Crous	KF442691	KF442658	–	–	–
	CBS 111045	Eucalyptus grandis	South Africa	M.J. Wingfield	KF442692	KF442659	–	–	–
	CBS 681.95	Eucalyptus bicostata	South Africa	M.J. Wingfield	KF442693	EU167579	–	–	–
Pallidocercospora heimii	CPC 11441	–	Brazil	A.C. Alfenas	KF442694	KF442660	–	–	–
	CPC 11716	–	Brazil	A.C. Alfenas	KF442695	KF442661	–	–	–
	CPC 11926	Acacia auriculiformis	Thailand	W. Himaman	KF442696	KF442662	–	–	–
	CPC 13099	Eucalyptus dunnii	Australia	A.J. Carnegie	KF442697	GQ852606	–	–	–
	CBS 110682	Eucalyptus sp.	Madagascar	P.W. Crous	KF442698	GQ852604	–	–	–
Pallidocercospora heimioides	CBS 111190	Eucalyptus sp.	Indonesia	M.J. Wingfield	KF442699	GU214439	–	–	–
	CBS 111364	–	Indonesia	M.J. Wingfield	KF442700	KF442663	–	–	–
Pallidocercospora irregulariramosa	CBS 111211	Eucalyptus saligna	South Africa	M.J. Wingfield	KF442701	KF442664	–	–	–
Pallidocercospora konae	CPC 10992	Eucalyptus sp.	Colombia	M.J. Wingfield	KF442702	KF442665	–	–	–
Pallidocercospora sp.	CPC 21817	Ventilago denticulata	Thailand	P.W. Crous	KF442703	KF442666	KF442645	–	–
Pallidocercospora thailandica	CBS 120723	Eucalyptus camaldulensis	Thailand	W. Himaman	KF442704	KF442667	–	–	–
Phaeophleospora stonei	CBS 120830	Eucalyptus sp.	Australia	P.W. Crous	KF442705	KF442668	–	–	–
Pseudocercospora angolensis	CBS 112748	Citrus sp.	Zimbabwe	P.W. Crous	JX902001	JX901878	–	–	–
	CBS 112933	Citrus sp.	Zimbabwe	M.C. Pretorius	JX902002	JX901879	–	–	–
	CBS 115645	Citrus sp.	Zimbabwe	P.W. Crous	JX902003	JX901880	–	–	–
	CBS 149.53	Citrus sinensis	Angola	T. de Carvalho & O. Mendes	JX902004	JX901881	–	–	–
	CBS 244.94	Citrus sp.	Zimbabwe	P.W. Crous	JX902000	JX901877	–	–	–
Pseudocercospora assamensis	CBS 122467	Musa sp.	India	I.W. Buddenhagen	JX902005	JX901882	–	–	–
Pseudocercospora atromarginalis	CPC 11372	Solanun nigrum	South Korea	H.D. Shin	JX902006	JX901883	–	–	–
Pseudocercospora basiramifera	CBS 111072	Eucalyptus pellita	Thailand	M.J. Wingfield	KF442706	GU253709	–	–	–
	CBS 114757	Eucalyptus pellita	Thailand	M.J. Wingfield	KF442707	GU253802	–	–	–
Pseudocercospora basitruncata	CBS 114664	Eucalyptus grandis	Colombia	M.J. Wingfield	KF442708	GU253710	–	–	–
Pseudocercospora cercidis-chinensis	CPC 14481	Cercis chinensis	South Korea	H.D. Shin	JX902007	JX901884	–	–	–
Pseudocercospora chiangmaiensis	CBS 123244	Eucalyptus camaldurensis	Thailand	R. Cheewangkoon	JX902008	JX901885	–	–	–
Pseudocercospora clematidis	CPC 11657	Clematis sp.	USA	M. Palm	JX902009	JX901886	–	–	–
Pseudocercospora crousii	CBS 119487	Eucalyptus sp.	New Zealand	C.F. Hill	KF442709	GQ852631	–	–	–
Pseudocercospora eucalyptorum	CBS 110776	Eucalyptus nitens	South Africa	P.W. Crous	KF442710	KF442669	–	–	–
	CBS 110903	Eucalyptus nitens	South Africa	P.W. Crous	KF442711	KF442670	–	–	–
	CBS 111268	Eucalyptus grandis	Kenya	M.J. Wingfield	KF442712	KF442671	–	–	–
Pseudocercospora flavomarginata	CBS 118824	Eucalyptus camaldulensis	China	M.J. Wingfield	JX902010	JX901887	–	–	–
	CBS 124990	Eucalyptus camaldulensis	Thailand	W. Himaman	JX902028	JX901905	–	–	–
Pseudocercospora fori	CBS 113285	Eucalyptus grandis	South Africa	G.C. Hunter	KF442713	GU253824	–	–	–
	CBS 113286	Eucalyptus sp.	South Africa	J. Roux	KF442714	KF442672	–	–	–
Pseudocercospora gracilis	CPC 11144	Eucalyptus sp.	Indonesia	M.J. Wingfield	JX902011	JX901888	–	–	–
	CPC 11181	Eucalyptus sp.	Indonesia	M.J. Wingfield	JX902012	JX901889	–	–	–
	CBS 111189	Eucalyptus urophylla	–	M.J. Wingfield	JX902013	JX901890	–	–	–
Pseudocercospora humuli-japonici	CPC 11315	Humulus japonicus	South Korea	H.D. Shin	JX902014	JX901891	–	–	–
Pseudocercospora madagascariensis	CBS 124155	Eucalyptus camaldulensis	Madagascar	M.J. Wingfield	JX902016	JX901893	–	–	–
Pseudocercospora marginalis	CBS 131582	Fraxinus rhynchophylla	South Korea	H.D. Shin	KF442715	GU253812	–	–	–
Pseudocercospora natalensis	CBS 111069	Eucalyptus nitens	South Africa	T. Coutinho	KF442716	KF302405	–	–	–
Pseudocercospora norchiensis	CBS 120738	Eucalyptus sp.	Italy	W. Gams	JX902017	JX901894	–	–	–
Pseudocercospora paraguayensis	CBS 111286	Eucalyptus nitens	Brazil	P.W. Crous	JX902018	JX901895	–	–	–
Pseudocercospora pini-densiflorae	CBS 125138	Pinus sp.	Japan	Sung-Oui Suh	JX902021	JX901898	–	–	–
	CBS 125140	Pinus kesiya	Japan	Sung-Oui Suh	JX902020	JX901897	–	–	–
Pseudocercospora pistacina	CPC 21874	Pistacia vera	Turkey	K. Sarpkaya	KF442719	KF442675	KF442648	KF442734	KF442638
	CPC 23117; 27NT080	Pistacia vera	Turkey	K. Sarpkaya	KF442717	KF442673	KF442646	KF442732	KF442636
	CPC 23118; 09mrk010	Pistacia vera	Turkey	K. Sarpkaya	KF442718	KF442674	KF442647	KF442733	KF442637
	CBS 135840; 45sln005	Pistacia vera	Turkey	K. Sarpkaya	KF442720	KF442676	KF442649	KF442735	KF442639
	CBS 135841; 63br043	Pistacia vera	Turkey	K. Sarpkaya	KF442721	KF442677	KF442650	KF442736	KF442640
Pseudocercospora plechranti	CPC 11462	Plectranthus	South Korea	H.D. Shin	JX902015	JX901892	–	–	–
Pseudocercospora pyracanthigena	CPC 10808	Pyracantha angustifolia	South Korea	H.D. Shin	KF252323	KF251823	–	–	–
Pseudocercospora rhoina	CPC 11464	Rhus chinensis	South Korea	H.D. Shin	JX902026	JX901903	–	–	–
Pseudocercospora robusta	CBS 111175	Eucalyptus robur	Malaysia	M.J. Wingfield	JX902027	JX901904	–	–	–
Pseudocercospora sphaerulinae	CBS 112621	Eucalyptus sp.	–	P.W. Crous	JX902029	JX901906	–	–	–
Pseudocercospora subulata	CBS 118489	Eucalyptus botryoides	New Zealand	M. Dick	JX902030	JX901907	–	–	–
Pseudocercospora tereticornis	CPC 13315	Eucalyptus nitens	Australia	P.W. Crous	JX902032	JX901909	–	–	–
	CBS 124996	Eucalyptus nitens	Australia	A.J. Carnegie	JX902033	JX901910	–	–	–
Pseudocercospora vitis	CPC 11595	Vitis vinifera	South Korea	H.D. Shin	JX902035	JX901912	–	–	–
Ramulispora sorghi	CBS 110578	Sorghum bicolor	South Africa	D. Nowell	KF442722	KF442678	–	–	–
	CBS 110579	Sorghum bicolor	South Africa	D. Nowell	KF442723	KF442679	–	–	–
Septoria astragali	CBS 109117	Astragalus glycyphyllos	Austria	G. Verkley	KF252350	KF251853	–	–	–
	CBS 123878	Astragalus glycyphyllos	Czech Republic	G. Verkley	KF252351	KF251854	–	–	–
Septoria cytisi	USO 378994	Laburnum anagyroides	Czech Republic	J. A. Baumler	–	JF700954	–	–	–
Septoria hibiscicola	CBS 128611	Hibiscus syriacus	South Korea	H.D. Shin	KF252430	KF251937	–	–	–
Septoria hippocastani	CBS 411.61	Aesculus hippocastanum	Germany	W. Gerlach	KF252432	KF251939	–	–	–
Septoria justiciae	CBS 128610	Justica procumbens	South Korea	H.D. Shin	KF252433	KF251940	–	–	–
Septoria lamiicola	CBS 109113	lamium album	Austria	G. Verkley	KF252443	KF251950	–	–	–
Septoria pistaciae	CBS 420.51	Pistacia vera	Italy	G. Goidánich	KF442724	KF252025	–	–	–
Septoria pistaciarum	CPC 23116; 5DMR032	Pistacia vera	Turkey	K. Sarpkaya	KF442725	KF442680	KF442651	KF442737	KF442635
	CPC 23114; 003c	Pistacia vera	Turkey	K. Sarpkaya	KF442726	KF442681	KF442652	KF442738	KF442641
	CPC 23115; 002B	Pistacia terebinthus	Turkey	K. Sarpkaya	KF442727	KF442682	KF442653	KF442739	KF442642
	CBS 135838; 45sln034	Pistacia vera	Turkey	K. Sarpkaya	KF442728	KF442683	KF442654	KF442740	KF442643
	CBS 135839; 001A	Pistacia vera	Turkey	K. Sarpkaya	KF442729	KF442684	KF442655	KF442741	KF442644
Septoria protearum	CBS 101013	Masdevallia sp.	Netherlands	W. Veenbaas-Rijks	KF252504	KF252013	–	–	–
	CBS 101354	Gevuina avellana	New Zealand	S. Ganev	KF252505	KF252014	–	–	–
	CBS 113392	Lobelia erinus	–	S. Wolcon	KF252507	KF252016	–	–	–
	CBS 410.61	Gerbera jamesonii	Italy	W. Gerlach	KF252514	KF252024	–	–	–
	CBS 566.88	Hedera helix	France	H.A. van der Aa	KF252515	KF442693	–	–	–
Septoria rumicum	CBS 503.76	Rumex acetosa	France	H. A. van der Aa	KF252523	KF252034	–	–	–
Septoria stellariae	CBS 102376	Stellaria media	Netherlands	G. Verkley	KF252567	KF252079	–	–	–
	CBS 102378	Castanea sativa	Netherlands	G. Verkley	KF252568	KF252080	–	–	–
Sonderhenia eucalypticola	CPC 11252	Eucalyptus globulus	Spain	M.J. Wingfield	KF442730	KF442685	–	–	–
Sphaerulina betulae	CBS 116724	Betula pubescens	Scotland	S. Green	KF252595	KF252107	–	–	–
	CBS 128600	Betula platyphylla	South Korea	H.D. Shin	KF252598	KF252110	–	–	–
Sphaerulina musiva	CBS 130559	Populus sp.	Canada	J. LeBoldus	KF252611	KF252124	–	–	–
	CBS 130562	Populus sp.	Canada	J. LeBoldus	KF252612	KF252125	–	–	–
Sphaerulina quercicola	CBS 109009	Quercus rubra	Netherlands	G. Verkley	KF252619	KF252132	–	–	–
	CBS 115016	Quercus robur	Netherlands	G. Verkley	KF252620	KF252133	–	–	–
Zymoseptoria verkleyi	CBS 133618	Poa annua	Netherlands	S.I.R. Videira	KF442731	KF442686	–	–	–

1CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; USO: United States Department of Agriculture, National Fungus Collections (BPI)

2Btub: β-tubulin; EF-1α: Translation elongation factor 1-alpha; ITS: internal transcribed spacers and intervening 5.8S nrDNA; LSU: 28S large subunit of the nrRNA gene; RPB2: RNA polymerase II second largest subunit.

DNA isolation, amplification and analyses

Genomic DNA was extracted from fungal colonies growing on MEA using the UltraCleanTM Microbial DNA Isolation Kit (MoBio Laboratories, Solana Beach, CA, USA) according to the manufacturer’s protocol. The primers LSU1Fd (Crous ) and LR5 (Vilgalys & Hester 1990) were used to amplify the partial 28S rRNA gene (LSU), ITS5 and ITS4 (White ) were used to amplify the ITS region T1 (O’Donnell & Cigelnik 1997) and b-Sandy-R (Stukenbrock ) were used to amplify the partial β-tubulin locus (Btub), EF1-728F (Carbone & Kohn 1999) and EF-2 (O’Donnell ) were used to amplify the partial translation elongation factor-1α locus (EF) and fRPB2-5F (Liu ) and fRPB2-414R (Quaedvlieg ) were used to amplify the partial RNA polymerase II second largest subunit locus (RPB2). A basic alignment of the obtained sequence data was first done using MAFFT v. 7 [(http://mafft.cbrc.jp/alignment/server/index.html) (Katoh )] and if necessary, manually improved in BioEdit v. 7.0.5.2 (Hall 1999). To check the congruency of the RPB2 and LSU datasets, a 70 % neighbour-joining (NJ) reciprocal bootstrap was performed (Mason-Gamer & Kellogg 1996, Lombard ). A Bayesian analysis (critical value for the topological convergence diagnostic set to 0.01) was performed on the concatenated LSU/RPB2 loci using MrBayes v. 3.2.1 (Huelsenbeck & Ronquist 2001) as described by Crous using nucleotide substitution models that were selected using MrModeltest v. 2.3 (Nylander 2004). Sequences derived from this study were lodged at GenBank, and the alignment was deposited in TreeBASE (www.treebase.org/treebase/index.html).

Morphology

Observations were made with a Zeiss V20 Discovery stereo-microscope, and with a Zeiss Axio Imager 2 light microscope using differential interference contrast (DIC) illumination and an AxioCam MRc5 camera and Zen software. Colony characters and pigment production were noted after 2 wk of growth on MEA, PDA and OA incubated at 25 °C. Colony colours (surface and reverse) were rated according to the colour charts of Rayner (1970). Morphological descriptions were based on cultures sporulating on PDA, and taxonomic novelties and metadata were deposited in MycoBank (www.MycoBank.org; Crous ).

RESULTS

The RPB2 and LSU sequence datasets did not show any conflicts in their tree topology for the 70 % reciprocal bootstrap trees, allowing us to combine them in the multigene analyses. The LSU sequence contained 745 base pairs, of which 99 where unique, the RPB2 sequence contained 317 base pairs, of which 157 where unique. For both datasets, the GTR-I-gamma substitution model (as calculated by MrModeltest) was used during the MrBayes run. During the generation of the tree (Fig. 1), a total of 7 216 trees were generated, and 5 412 (75 %) of them where sampled for the final tree

Fig. 1

A Bayesian 50 % majority rule RPB2/LSU consensus tree containing representative isolates belonging to Pseudocercospora and related genera (Mycosphaerellaceae). Bayesian posterior probabilities support values for the respective nodes are displayed in the tree. A stop rule (set to 0.01) for the critical value for the topological convergence diagnostic was used for the Bayesian analysis. The tree was rooted to Zymoseptoria verkleyi (CBS 133618). The scalebar indicates 0.1 expected changes per site.

TAXONOMY

Cylindroseptoria pistaciae Quaedvlieg et al., Stud. Mycol. 75: 359 (2013).

(Fig. 2)

Fig. 2

Cylindroseptoria pistaciae (CBS 471.69). A–B. Conidiomata sporulating in culture. C–D. Intercalary chains of chlamydospore-like cells. E–F. Conidiogenous cells. G–H. Conidia. Bars = 10 μm, H applies to C and D.

Description: Conidiomata pycnidial, erumpent, globose, black, separate, with a black crusty outer layer of cells, to 200 μm diam, with a central ostiole; wall of 3–6 layers of brown textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells phialidic (mostly monophialidic, but a few observed to also be polyphialidic), lining the inner cavity, hyaline, smooth, ampulliform, 5–8 × 3–4 μm, proliferating percurrently (inconspicuous) or with periclinal thickening at apex (also occurring as solitary loci on superficial hyphae surrounding pycnidia). Conidia hyaline, smooth, cylindrical, mostly straight, rarely slightly curved, apex subobtuse, base truncate, guttulate, aseptate, (9–)11–13(–18) × 2.5–3(–3.5) μm (from Quaedvlieg )

Culture characteristics: Colonies on PDA flat, circular, lacking aerial mycelium, surface fuscous-black, reverse olivaceous-black, after 14 d at 24 °C, 3.5 cm diam; on MEA surface fuscous-black, reverse olivaceous-black, after 14 d, 4.5 cm diam; on OA similar to PDA.

Type: Spain: Mallorca: El Arenal, on leaves of Pistacia lentiscus, 25 May 1969, H. A. van der Aa (CBS H-21301 – holotype; culture ex-type CBS 471.69).

Notes: Quaedvlieg established the genus Cylindroseptoria for taxa having cupulate to pycnidial conidiomata, and phialidic conidiogenous cells with periclinal thickening, that give rise to cylindrical, aseptate conidia. Although Cylindroseptoria pistaciae was introduced as a novel species (from symptomatic leaves of Pistacia lentiscus), no information is available about its potential role as plant pathogen.

Pseudocercospora pistacina (Allesch.) Crous, Quaedvlieg & Sarpkaya, comb. nov.

Basionym: Septoria pistacina Allesch., Rabenh. Krypt.-Fl. 1(6): 830 (1900) [“1899”].

Synonyms: Dothidea pistaciae Lév., in Démidoff, Voy. Russ. Mér. 2: 108 (1842).

Non Pseudocercospora pistaciae (Chupp) Crous & U. Braun, Mycotaxon 78: 338 (2001).

Septoria pistaciae (Lév.) Cooke, Grevillea 13 (66): 45 (1884); nom. illegit., non S. pistaciae Desm. 1842.

Mycosphaerella pistacina Chitzan., Ann. Inst. Phytopath. Benaki 10: 42 (1956).

MycoBank MB805893

(Fig. 3)

Fig. 3

Pseudocercospora pistacina (CBS 135840). A. Fruit tree orchard. B–C. Leaf spots. D. Disease symptoms on fruit. E. Conidia cirrhi oozing from immersed pycnidial conidiomata. F–G. Vertical section through pycnidia. H. Conidiogenous cells giving rise to conidia. I. Conidia. Bars: F = 300 μm, G = 150 μm, H–I = 10 μm.

Description: Leaf spots numerous, brown, amphigenous, angular, confined by leaf veins, to 30 mm long, 3–6 mm diam, containing numerous small, aggregated, immersed conidiomata. Fruit spots grey to pale brown, 1–4 mm diam, coalescing to form larger spots, surrounded by a distinct, reddish margin. Conidiomata subepidermal, globose to depressed, to 300 μm diam with a wide central ostiole, to 100 μm diam; wall 10–20 μm thick, of 3–6 layers of brown textura angularis. Conidiophores subcylindrical, pale brown, smooth, 0–3-septate, branched or not, 10–30 × 3–5 μm. Conidiogenous cells terminal and sublateral, pale brown, smooth, subcylindrical to doliiform, 6–15 × 2.5–4 μm; proliferating several times percurrently at the apex. Conidia pale brown, smooth, guttulate, subcylindrical, curved, medianly 1-septate, constricted at the septum, apex obtuse, tapering at the base to a truncate hilum, 1.5–2 μm diam, (32–) 35–42(–50) × (3–)3.5–4(–5) μm.

Chitzanidis (1956) reports ascomata as 90–110 × 80–110 μm, asci as 44.5–54.5 × 13–14.5 μm, and ascospores as 26–40 × 3–5 μm.

Culture characteristics: Colonies after 2 wk at 24 °C reaching 10 mm diam, erumpent with sparse aerial mycelium and even, lobate margins; on OA, MEA and PDA dirty white, remaining sterile; in reverse iron-grey.

Specimens examined: Turkey: Manisa: Selendi, on Pistachio vera, 2010, K. Sarpkaya (CPC 45sln005 = CBS 135840). Gaziantep: Nizip, on P. vera, 2010, K. Sarpkaya (CPC 27NZ080 = CPC 23117). Sanliurfa: Birecik, on P. vera, 2010, K. Sarpkaya (CPC 63br043 = CBS 135841). Aydin: Merkez, on P. vera, 2010, K. Sarpkaya (CPC 09mrk010 = CPC 23118); collection site unknown, on P. vera, 2010, K. Sarpkaya (CPC 21874).

Notes: Because of the pycnidial conidiomata and pigmented conidia, Pseudocercospora pistacina can be confused with Phaeophloeospora or Kirramyces (syn. Teratosphaeria; Crous , b), though it is phylogenetically unrelated to these genera. Pseudocercospora pistacina clusters basally within Pseudocercospora, but based on the genes studied here, could not be recognised as a separate genus. The genus Pseudocercospora was recently circumscribed as having species with conidiophores that are solitary, fasciculate, synnematal, or arranged in sporodochia, giving rise to conidia that are pigmented, have unthickened or slightly thickened and darkened scars (Crous ). By including Septoria pistacina in Pseudocercospora, we are expanding the generic circumscription of the latter to also include taxa with well-defined pycnidial conidiomata (on host and in culture). Conidiomatal structure has to date been paramount in identifying taxa with enclosed conidiomata (Sutton 1980, Nag Raj 1993), and thus P. pistacina is rather atypical within Pseudocercospora s. str.

Septoria pistaciae Desm., Annls Sci. Nat., Bot., sér. 2 17: 112 (1842).

Synonyms: Phloeospora pistaciae (Desm.) Petr., Annls mycol. 20: 18 (1922).

Cylindrosporium pistaciae (Desm.) Vassil., Fungi Imp. Paras. 2: 510 (1950).

(Fig. 4)

Fig. 4

Septoria pistaciae (PC 0142143). A. Herbarium specimen. B–F. Conidiogenous cells giving rise to conidia. G. Conidia. Bars = 10 μm, D applies to E–G.

Description: Leaf spots initially small, orbicular or oblong, scattered, brown to dark brown, 1–2 mm diam, becoming irregular, 5–10 mm, covering large portions of leaf, becoming greyish brown with distinct, narrow brown margin. Conidiomata pycnidial, amphigenous, separate or densely aggregated in the centre of leaf spots, immersed, becoming erumpent, brown to dark brown, globose to pyriform, (40–) 70–90(–120) μm diam, with central ostiole, 15–20 μm diam; wall of 3–4 layers of brown textura angularis. Conidiophores reduced to conidiogenous cells, or up to 4-septate, subcylindrical with lateral and terminal conidiogenous cells, 5–25 × 3–4 μm. Conidiogenous cells hyaline, smooth, ampulliform to subcylindrical, 5–10 × 3–4 μm, lining inner layer of conidiomatal cavity, proliferating sympodially, rarely percurrently. Conidia hyaline, smooth, 0–3-septate, (9–)13–22(–25) × (1.5–)2(–3) μm, obclavate to narrowly subcylindrical, apex subobtuse, base obconically truncate with flattened scar. Spermatial state occurring in conidiomata along with conidia. Spermatogenous cells hyaline, smooth, ampulliform, 4–6 × 3–5 μm. Spermatia hyaline, smooth, ellipsoid to subcylindrical, with obtuse ends, 2.5–3.5 × 1.5 μm.

Culture characteristics: Colonies after 2 wk at 25 °C reaching 40 mm diam on OA; surface sienna, smooth, with even margins, lacking aerial mycelium; culture sterile on OA, PDA, MEA and on barley leaves placed on synthetic nutrient-poor agar (Crous ).

Specimens examined: France: on leaves of Pistacia vera, 1842, Desmazière [Pl. Crypt. Nord Fr., fasc. 24, no. 1181] (PC0142144 – holotype; authentic specimen from general herbarium (PC) no. 1181, PC0142143). – Italy: on leaves of P. vera, June 1951, G. Goidánich (CBS 420.51; culture sterile).

Notes: Septoria pistaciae is part of the species complex for which Verkley adopted the oldest name, S. protearum, which has an ex-type culture. Isolates in this complex could not be robustly distinguished based on a seven-gene phylogeny, and represent collections with a range of hosts covering six different plant families. Whether this is one plurivorous taxon that can undergo host jumping (Crous & Groenewald 2005), or several closely related taxa that cannot be distinguished based on the set of genes employed by Verkley , awaits further study and inoculation trials.

Andrianova & Minter (2004) described conidia of S. pistaciae as 1–3-septate, (20–)22–25(–34) × 1.5(–2) μm, obclavate to narrowly subcylindrical (based on type material, LE 42353). Our measurements from type material are considerably smaller, namely 0–3-septate, (9–)13–22(–25) × (1.5–)2(–3) μm (PC 0142144). Type material of S. protearum has conidia that are (0–)1–3(–4)-septate, (6–)12–22(–30) × 1.5–2 μm, obclavate to narrowly subcylindrical (Swart , Crous ). If these two taxa are eventually shown to be synonymous, the name S. pistaciae (1842) predates that of S. protearum (1998), but even that may not be the oldest epithet for this taxon. The single isolate available to us for study (CBS 420.51) proved to be sterile, so its morphology could not be confirmed.

Septoria pistaciarum Caracc., Boll. Stud. Inform. R. Giard Colon Palermo 13: 10 [extr.] (1934).

Synonym: Mycosphaerella pistaciarum Chitzan., Ann. Inst. Phytopath. Benaki 10: 42 (1956).

(Fig. 5)

Fig. 5

Septoria pistaciarum (CBS 135838). A. Colony sporulating on SNA with sterile barley leaves. B. Colony on OA. C–E. Conidiogenous cells giving rise to conidia. F–G. Conidia. Bars: A–B = 200 μm, C, F–G = 10 μm, C applies to D and E.

Description: Leaf spots angular, brown, amphigenous, 1–2 mm diam, coalescing to become larger leaf spots, confined by leaf veins. Conidiomata pycnidial, erumpent, brown, globose, to 200 μm diam, with central ostiole, exuding a crystalline cirrhus of conidia; wall of 3–6 layers of brown textura angularis. Conidiophores reduced to conidiogenous cells, or one supporting cell which can be branched at the base. Conidiogenous cells lining the inner cavity, hyaline, smooth, subcylindrical to ampulliform or doliiform, 5–15 × 2.5–4 μm, proliferating percurrently near apex, or sympodially. Conidia solitary, hyaline, smooth, guttulate, straight to curved, narrowly obclavate to subcylindrical, (1–)3-septate, apex subobtuse, base obconically truncate, 2 μm diam with minute marginal frill, (45–)55–65(–75) × (2.5–)3(–3.5) μm.

Chitzanidis (1956) reports ascomata as 95–130 × 85–120 μm, asci 47.5–60.5 × 8–12 μm, and ascospores 18–30 × 3–5 μm.

Culture characteristics: Colonies after 2 wk at 24 °C reaching 30 mm diam. Colonies erumpent, folded with feathery, lobate margins. On PDA surface olivaceous grey with patches of pale olivaceous grey and dirty white, reverse olivaceous grey. On OA surface greyish sepia with patches of dirty white, and an umber pigment diffusing into agar. On MEA surface pale olivaceous grey with patches of dirty white; olivaceous grey in reverse.

Specimens examined: Turkey: Hatay: Merkez, on Pistacia terebinthus, 2012, K. Sarpkaya (CPC 002B = CPC 23115). Sakarya: Geyve, on P. vera, 2012, K. Sarpkaya (CPC 003C = CPC 23114). Kutahya: Emet, on P. vera, 2012, K. Sarpkaya (CPC 001A = CBS 135839). Manisa: Selendi, on P. vera, 2012, K. Sarpkaya (CPC 45sln034 = CBS 135838); Demirci, on P. vera, 2012, K. Sarpkaya (CPC 5DMR032 = CPC 23116).

Notes: Septoria pistaciarum is morphologically distinct from the other species occurring on pistachio, in having much larger conidia (45–75 × 2.5–3.5 μm). In the field it can also be disnguished on diseased host plants in causing more angular leaf spots, confined by leaf veins.

DISCUSSION

The aim of the present study was to clarify which species of Septoria occur on pistachio, and to place them in a phylogenetic context within Mycosphaerellaceae, as recently circumscribed (Quaedvlieg , Verkley ). From results obtained, it is clear that up to four septoria-like taxa occur on pistachio, of which two belong to other genera, namely Cylindroseptoria pistaciae and Pseudocercospora pistacina. The remaining two species represent true species of Septoria, namely S. pistaciae and S. pistaciarum. Because of discrepancies in previously published literature, much confusion arose regarding how to distinguish these taxa. In the present study we have been able to compile a key to facilitate identification of these taxa (see below). Sexual morphs have also been described for two of these taxa, namely Septoria pistacina (i.e. Mycosphaerella pistacina) and Septoria pistaciarum (i.e. Mycosphaerella pistaciarum) (Chitzanidis 1956, Teviotdale ). However, because the genus Mycosphaerella is restricted to Ramularia (Verkley , Crous , b, Koike ), in moving towards a single nomenclature for fungi (see Hawksworth , Wingfield ), the use of Mycosphaerella should be avoided for the mycosphaerella-like sexual morphs linked to Septoria.

The placement of Septoria pistacina in Pseudocercospora is somewhat controvertial, as it has a typical pycnidial conidioma, rather than superficial fascicles or synnemata encountered in Pseudocercospora. Phylogenetically, however, there is no support for recognising S. pistacina as a separate genus based on it being a “pigmented Septoria”. Morphologically, the conidiogenous cells and conidia fit the circumscription of Pseudocercospora, but the conidiomatal anatomy does not. Although species of Capnodiales are known to have synasexual morphs with closed and open conidiomata (Crous , 2009a, b), this is the first example of a taxon with a pycnidial conidioma that clusters among species with fasciculate conidiomata. In addition to S. pistacina, we are also aware of a second as yet undescribed species of “pigmented Septoria” (Crous et al., unpubl.), which again clusters in Pallidocercospora (Crous ). Another example of a genus reported to have acervuli, but observed to have superficial conidiomatal fascicles, is Ciferiella, which has also been reduced to synonymy with Pseudocercospora (Quaedvlieg ). These findings support the view that conidiomatal morphology in Pseudocercospora is a continuum from sporulating superficially (fascicles, synnemata, sporodochia), via acervuli, to sporulation in an enclosed structure (pycnidia).

In spite of being morphologically distinct, that two of the reported septoria-like taxa on pistachio represent different genera is rather suprising. Although the pathological relevance of Pseudocercospora pistacina (as S. pistacina), S. pistaciae, and S. pistaciarum on Pistacia vera is well-documented (Michailides 2005), nothing is known about that of Cylindroseptoria pistaciae, other than it was associated with leaf spots of Pistacia lentiscus in Spain. Furthermore, it also appears that the importance of certain diseases of pistachio differs between regions or countries. Approximately 85 % of the world’s pistachio production presently comes from Iran, the USA, and Turkey (http://faostat.fao.org/site/339/default.aspx). However, pistachio is irrigated in Iran and the USA, where more rounded fruit bearing cultivars are grown. In contrast, irrigation is not practiced in Turkey, and more elongated fruit cultivars are commonly grown there. These differences in cultivation practices also lead to differences in phytopatological problems. In the USA, the main pathogens are reported to be Botryosphaeria dothidea, Botrytis cinerea, and Alternaria alternata. In Turkey, however, Pseudocercospora pistacina is the most common disease of pistachio (Dinç 1983, Michailides ), leading to reports of 3–100 % yield loss in epidemic years by this pathogen (Dinç ).

Conidia aseptate, 9–18 × 2.5–3.5 μm ....................

Conidia septate .................... 2

Conidia pale brown, medianly 1-septate, 32–50 × 3–5 μm ....................

Conidia hyaline, 1–3-septate ..................... 3

Conidia 9–34 × 1.5–3 μm ....................

Conidia 45–75 × 2.5–3.5 μm ....................