Characterization of Phoma adonidicola causing a spot blighton Adonis palaestina

Yan Li & Chunsheng Zheng & Zhihui Zhao &

Guozhong Lu & Xuechi Fu & Ruhong Mei &Jinqin Li & Qi Wang

Accepted: 25 April 2013 /Published online: 22 June 2013# KNPV 2013

Abstract Adonis palaestina Boiss. is one of the topthree natural sources of red pigment astaxanthin, whichhas been used as a valuable antioxidant nutraceuticaland a feed additive for salmonid fish raising. Since2004, a blight disease causing significant damage toplants of A. palaestina in Inner Mongolia, China hasoccurred. The disease caused small, brown lesions onpetioles and stems of the host plants. The disease ini-tially appeared in the field as a brown necrosis on lowerparts of the plant, and eventually expanded to the wholeplant resulting in complete defoliation. The fungus con-sistently isolated from symptomatic tissues was identi-fied as Phoma adonidicola based on morphological

characteristics. Phylogenetic analysis based on LSU(Large Subunit - 28S), ITS (ITS1-5.8S-ITS2 region),and TUB (β-tubulin) showed that the P. adonidicolaisolates fall in the same well-supported clade, whichis closely related to Stagonosporopsis ajacis. Allisolates of P. adonidicola also caused typical spotson inoculated plants, and were successfully reisolatedfrom the symptomatic tissues. The disease of A.palaestina was proposed as spot blight.

Keywords Spot blight . Koch’s postulates .Multigeneanalysis .Adonis palaestina

Introduction

Adonis palaestina Boiss. (Ranunculaceae), a diploidannual herbaceous plant originated from Palestine,Israel, Lebanon, Jordan, and Syria, is rich in astaxanthinespecially in petals of its bright red flowers (Wang 1994;Rayton et al. 2006). The red carotenoid pigment astax-anthin is a powerful antioxidant used in the nutraceuticalindustry (Fassett and Coombes 2009; Higuera-Ciapara etal. 2006; Pashkow et al. 2008) and also used as a fish-food additive in aquaculture as it imparts the desirablepink colouration to farmed salmonids (e.g. salmon, trout)and crustacea (e.g. shrimp) (Rayton et al. 2006; Seyboldand Goodwin 1959). The commercial pigment is mainlysynthesized by chemical methods, and partially extractedfrom some natural sources, such as crustaceans (López etal. 2004), the yeast Phaffia rhodozyma (Johnson et al.

Eur J Plant Pathol (2013) 137:127–136DOI 10.1007/s10658-013-0224-5

Y. Li :X. Fu : R. Mei :Q. Wang (*)Key Laboratory of Plant Pathology,Ministry of Agriculture, Department of Plant Pathology,China Agricultural University,No. 2 Yuanmingyuan West Road,Beijing 100193, Chinae-mail: wangqi@cau.edu.cn

C. ZhengBeijing Capital International Airport Entry-ExitInspection and Quarantine Bureau,Beijing 100621, China

Z. Zhao :G. LuCollege of Environment and Resources,Dalian Nationalities University,Dalian 116600, China

J. LiTongliao Academy of Agricultural Science,Inner Mongolia 028015, China

1980; Sedmak et al. 1990), and green algae likeHaematococcus pluvialis (Kobayashi et al. 1991;Olaizola 2000). Extraction of astaxanthin from A. palae-stina is inexpensive and avoids accumulation or pollu-tion with chemical by-products, which makes the plant avaluable natural resource (Rayton et al. 2006). A. palae-stina has been widely grown in Inner Mongolia, Chinasince it was introduced for the production of astaxanthinin 2004 (Zhang et al. 2004).

In summer of 2004, a disease causing spots onleaves, stems and petioles was observed in fields inTongliao, Inner Mongolia, China, during investigationof plant diseases on A. palaestina. These spots gener-ally coalesced to cause blight on above-ground tissuesand was presumably associated with the fungusPhoma adonidicola (Li et al. 2006). The disease hasbeen found to be serious for many years, and itcaused up to 60 % loss in harvest of A. palaestina,imposing a direct threat to its sustainable produc-tion. This paper was aimed at characterization ofthe pathogen and setting up a base for scientificcontrol of the disease.

Materials and methods

Isolation of the causal agent

Following in-field observation of disease develop-ment, five to seven diseased plants were randomlyselected from five commercial fields in the Tongliao,Inner Mongolia, China. Typically symptomatic stems,roots and petioles were surface-sterilized by soaking in1 % sodium hypochlorite solution for 5 min and thenrinsing three times with sterile distilled water. Smallpieces of tissues (ca 5×5 mm) were aseptically cutfrom margins of the lesions and placed on potato-dextrose agar (PDA) (200 g potatoes, 20 g dextrose,20 g agar/1 l distilled water) containing 100 μg ml−1

streptomycin sulphate (Den Breeÿen et al. 2006). Theplates were incubated in dark at 26–28 °C. After 3 to5 days of incubation, the fungal colonies were trans-ferred to fresh PDA plates and incubated at 26–28 °C.The fungal isolates were subcultured for purificationby means of single spores and then subsequentlytransferred into PDA slants for storage at 4 °C.Mycelia from the purified fungal colony were suspendedin 15 % glycerol in cryovials and frozen at −80 °C forlong-term storage.

Pathogenicity test

The fungal isolates were grown on PDA for 7 days at22 °C in dark. Sporulation was induced by transferringa 5-mm mycelial plug onto oatmeal agar (OA) (15 g ofoatmeal, 20 g agar/1 l distilled water) (Den Breeÿen etal. 2006). These plates were incubated under near UVradiation with a 12-h photoperiod at 22 °C for 8 days(Boerema et al. 2004). Conidial suspensions wereprepared by flooding the plates with 5 ml of steriledistilled water. The conidia were collected by pouringthe suspension through sterile lens wiping papers. Thenumber of conidia was counted under a haemacytom-eter and adjusted to approximately 5×106 spores ml−1

with sterile distilled water.The A. palaestina seedlings used for inoculation

were grown from seeds, which were disinfected with1 % sodium hypochlorite solution for 10 min andplanted into pots containing a 2:1:1 soil/peat/vermicu-lite mixture sterilized with dry heat at 170 °C for 2 h.The pathogenicity test was performed in a dew cham-ber (22~24 °C, >70 % RH). Twenty-eight fungalisolates originating from various plant parts (stem,petiole and root; Table 1), locations and growingstages were selected for the pathogenicity assay.Four-month-old plants were uniformly misted untilrun-off with a spore suspension containing Tween 20(0.025 %). Each experiment was performed in tripli-cate. Each replicate was made using 10 plants and thecontrol plants were sprayed with sterile distilled water.The inoculated seedlings were covered with polyethyl-ene bags for 48 h to create moist conditions and incu-bated in the chamber under a 12 h photoperiod.

Four to eight days after incubation evaluations fordisease severity were made. Plants were rated for dis-ease severity based on the percentage of surface areainfected. Each plant was rated on a scale of 0 to 5, where0=no symptom, 1=less than 10 % of the plant surfacecovered by lesions, 2=10 to 25 %, 3=25 to 50 %, 4=50to 75 % and 5=75 to 100 % resulting in plantdeath. A disease severity index (DSI) was calculatedaccording to the following formula: DSI ¼ 100�P

ratings of each plantð Þ Maximal grade� numberð=of plants ratedÞ: (Cober et al. 2003; Sharma andKolte 1994). This results in a DSI of 0 for no plantsrated to be infected and a DSI of 100 for all ratedplants killed by the disease. Analysis of variancewas performed on DSI data with the program SAS(Cary, North Carolina). The pathogen was re-isolated

128 Eur J Plant Pathol (2013) 137:127–136

from the artificially inoculated plants after showingtypical symptoms of the disease.

Identification of the casual agent

The Phoma isolates were firstly examined morpholog-ically. For this purpose, they were grown on OA indark for 7 days under near-ultraviolet radiation with a12-h photoperiod at 22 °C (Boerema et al. 2004). Thecultures after 7 and 14 days were investigated forcolony diameter, pigmentation, conidial morphologyand diffusible antibiotic metabolite ‘E’ (Boerema et al.2004), which was tested by placing a drop of concen-trated NaOH on edge of the colonies on OA to observethe colour variation.

For phylogenetic analysis, genomic DNAs of 12Phoma isolates were extracted from mycelia by usingFastDNA kit and FastPrep FP-24 instrument (MPBiomedicals Inc., Irvine, CA) according to the manufac-turer’s instructions. TheDNA sequences for three nuclearregions were analyzed: LSU (Large Subunit - 28S), ITS(ITS1-5.8S-ITS2 region), and TUB (β-tubulin). TheLSU region was amplified with the primers LR0R(Rehner and Samuels 1994) and LR7 (Vilgalys andHester 1990). The ITS region of the nuclear ribosomalDNAwas amplified with the primers ITS1 (Gardes andBruns 1993) and ITS4 (White et al. 1990). A part of theTUB gene was amplified with primers TUB2Fd andTUB4Rd (Aveskamp et al. 2009). PCRs were performedin aMyCycler™ Thermal Cycler (BIO-RAD, California,US) in a total volume of 50 μl with 5 μl of EasyTaqBuffer (10×) (TansGen Biotech, Beijing, China), 4 μl ofdNTPs (2.5 mM), 1 μl of each primer (10 μM) (BeijingSunbiotech Co., Ltd., Beijing, China), 0.5 μl (1 U per50 μl) of EasyTaq DNA polymerase (TansGen Biotech,Beijing, China), 10 to 50 ng of DNA template, and36.5 μl of highly purified sterile H2O. In all PCR sets, anegative control without DNAwas included.

Cycling parameters for all three regions were 5min at95 °C, followed by 35 cycles of 30 s at 95 °C, annealing(see below), 2 min at 72 °C, followed by 7 min at 72 °C.The annealing steps were performed for 45 s at 48 °C forLSU, 50 s at 48 °C for ITS, and 30 s at 52 °C for TUB,respectively. The PCR products were analyzed by elec-trophoresis on a 1 % (w/v) agarose gel containing0.1 mg ml−1 ethidium bromide in 1× TAE buffer(0.4 M Tris, 0.05 M NaAc, 0.01 M EDTA, pH 7.85)and visualized under UV light. Trans 2K Plus II(TansGen Biotech, Beijing, China) was applied as sizestandard. PCR products were purified and sequenced inboth directions using the same primer combinations byBeijing Biomed Co., Ltd (Beijing, China). Sequences

Table 1 Source and ability of the fungal isolates included inthis study to cause stem blight of Adonis palaestina in a growthchamber

Isolatea Genus Source tissue Disease severityindexb

4 days 8 days

LY01 Phoma Stem 46.96 92.00

LY02 Phoma Stem 78.44 97.33

LY03 Phoma Petiole 100.00 100.00

LY55 Phoma Stem 92.00 96.00

LY59 Phoma Root 88.67 99.33

LY62 Phoma Petiole 54.91 72.07

LY64 Phoma Petiole 86.22 98.67

LY67 Phoma Stem 92.67 98.00

LY69 Phoma Root 69.33 96.00

LY70 Phoma Root 75.11 90.89

LY71 Phoma Stem 84.67 97.33

LY72 Phoma Stem 67.09 98.67

LY73 Phoma Stem 67.64 98.67

LY77 Phoma Stem 88.67 99.33

LY82 Phoma Petiole 50.91 86.00

LY06 Plectosporium Stem 20.83 21.67

LY07 Plectosporium Stem 17.5 18.33

LY10 Plectosporium Root 14.17 17.5

LY14 Plectosporium Stem 22.00 22.42

LY15 Plectosporium Stem 44.00 40.00

LY06 Fusarium Stem 0.00 0.00

LY84 Fusarium Root 0.00 0.00

LY04 Colletotrichum Petiole 0.00 0.00

LY05 Colletotrichum Stem 0.00 0.00

LY24 Alternaria Root 0.00 0.00

LY30 Alternaria Stem 0.00 0.00

LY35 Alternaria Stem 0.00 0.00

LY37 Alternaria Petiole 0.00 0.00

a Isolate LY62 and LY69 are the isotype strain IBE 002002 and002003, respectively, mentioned in Li et al. (2006)bDSI refer to Disease Severity Index, andwas calculated accordingto the following formula: DSI ¼ 100�P

ratings of eachðplantÞ Maximal grade� number of plants ratedð Þ= (Cober etal. 2003; Sharma and Kolte 1994). Plants were rated for diseaseseverity based on the percentage of surface area infected. Eachplant was rated on a scale of 0 to 5, where 0=no symptoms, 1=lessthan 10 % of the plant surface was covered by lesions, 2=10 to25%, 3=25 to 50%, 4=50 to 75% and 5=75 to 100% resulting inplant death

Eur J Plant Pathol (2013) 137:127–136 129

were assembled and edited with Gap4 using theSTADEN package (http://staden.sourceforge.net/).Ambiguous regions on both sides were excluded fromthe analyses.

Sequence queries were submitted to the NationalCenter for Biotechnology Information (NCBI) BlastNetwork Server (BlastN 2.2.18). Maximum parsimony(MP) analyses were firstly performed on individual genedatasets using Mega 4.0 software (Tamura et al. 2007).Once topological concordance was established amongthe three datasets, theywere then analyzed as a combineddataset with relative branch supports obtained with 1,000bootstrap replicates. Phylogenetic analyses also includedthe data sets produced by Aveskamp et al. (2010) andVaghefi et al. (2012) that were obtained from GenBank(Table 2). The sequences ofAscochyta hordei var. hordei(CBS 544.74) and Phoma paspali (CBS 560.81 & CBS561.81) were used for outgroup purposes.

Results

Disease symptoms and fungal isolates

The disease initially appeared on plants in late May orearly June when daily temperature reached 20–25 °C andrainfall occurred. The typical symptoms began withsmall, yellow-brown lesions on stem bases and lowerpetioles near soil (Fig. 1a). Over time, the lesions grad-ually enlarged, coalesced, and later became necrotic andblack-brown and covered most parts of the stems andpetioles (Fig. 1b). With the plant growing, the lesionsspread to the upper parts of the plants (Fig. 1c). Whenroots of the diseased plants were longitudinally dissected,dark-brown stripes were observed in the central vasculartissues of some diseased plants (Fig. 1d). As the diseasedeveloped, the lower leaves of the diseased plants grad-ually turned into dark brown in colour, and cracked afterdrying under drought conditions (Fig. 1e). The seriouslyinfected leaves defoliated earlier only with stems left(Fig. 1f). When the diseased tissues were incubated onmoist filter paper in Petri dishes, abundant pycnidiatypical of Phoma were observed after days. The diseasewas therefore named spot blight.

In total 226 isolates were obtained from the diseasedsamples collected in the field. Of these isolates, 148displayed typical morphology of Phoma, which wasconsistently isolated from stems, petioles and roots ofthe diseased plants (Fig. 2). Moreover, Phoma sp. had

the highest isolation frequency from stems and petiolesfollowed by roots (Fig. 2). Besides Phoma, 28 isolatesof Fusarium, 24 isolates of Plectosporium, 24 isolatesof Alternaria, and two isolates of Colletotrichumwere also recovered from the diseased tissue(Fig. 2). Twenty-eight isolates (15 Phoma, twoFusarium, five Plectosporium, four Alternaria, andtwo Colletotrichum; Table 1) originating from variousparts (stem, petiole or root), locations and growingstages were selected for the pathogenicity assay.

Pathogenicity test

All the Phoma isolates caused typical lesions oninoculated A. palaestina plants 2–3 days after inocula-tion (Table 1) and the symptoms were similar to thoseobserved in the field (Fig. 3a). Pycnidia were observedon stems 5–6 days after inoculation (Fig. 3b). The rootvascular tissues of some inoculated plants became darkbrown. We also noted that the five isolates ofPlectosporium sp. caused small brownish lesions onstems and petioles (Fig. 3c, Table 1). However, theselesions were different in appearance from those causedby the Phoma isolates and no pycnidia were observedon the inoculated plants. None of the Fusarium,Alternaria or Colletotrichum isolates caused any symp-toms on the inoculated plants (Table 1). No diseasesymptom was observed on control plants (Table 1). P.adonidicola was re-isolated from symptomatic tissuesof the artificially inoculated plants. These resultsindicated that Phoma was responsible for the spotblight symptoms on A. palaestina.

Identification of the casual agent

Based on culture growth, pigment formation, and char-acteristics of pycnidia, conidia and chlamydospores, allPhoma isolates from A. palaestina showed typical fea-tures of Phoma adonidicolaYan Li, Q.Wang&G. Z. Lu(Li et al. 2006). The morphological identification resultofP. adonidicolawas further confirmed by themultigeneanalysis. The sequences of LSU (GenBank Accessionnumbers JQ934854-JQ934865), ITS (GenBankAccession numbers JQ934830-JQ934841) or TUB(GenBank Accession numbers JQ934842-JQ934853)of the examined Phoma isolates were most similar tothose of Stagonosporopsis ajacis (synonym P. ajacis)strain CBS 176.93 and CBS177.93 and S. valerianellae(synonym P. valerianellae) strain CBS 273.92 and CBS

130 Eur J Plant Pathol (2013) 137:127–136

Table 2 Names, sources, origin, and sequence accession numbers of reference strains

Strain no.a Holomorphb GenBank accession numbers Host Origin

LSU ITS TUB

CBS 544.74 Ascochyta hordeivar. hordei

EU754134 GU237887 GU237488 Triticum aevestum South Africa

CBS 101150; PD 79/118 Boeremia exiguavar. exigua

GU237933 GU237715 GU237495 Cichorium intybus Netherlands

CBS 431.74; PD 74/2447 Boeremia exiguavar. exigua B

EU754183 FJ427001 FJ427112 Solanum tuberosum Netherlands

CBS 125.82; IMI 1331914;CECT 20044

Epicoccum nigrum GU237974 FJ426995 FJ427106 Human Netherlands

CBS 173.73; ATCC 24428;IMI 164070

Epicoccum nigrum T GU237975 FJ426996 FJ427107 Dactylis glomerata U.S.A.

CBS 107.96; PD 73/598 Phoma aquilegiicola B GU238041 GU237735 GU237582 Aconitum pyramidale Netherlands

CBS 108.96; PD 79/611 Phoma aquilegiicola B GU238042 GU237736 GU237583 Aquilegia sp. Netherlands

CBS 109179; PD 90/835-1 Phoma digitalis GU238066 GU237744 GU237604 Digitalis sp. Netherlands

CBS 229.79; LEV 7660 Phoma digitalis B GU238067 GU237802 GU237605 Digitalis purpurea New Zealand

CBS 123394 Phoma infossa GU238088 FJ427024 FJ427134 Fraxinuspennsylvanica

Argentina

CBS 123395 Phoma infossa T GU238089 FJ427025 FJ427135 Fraxinuspennsylvanica

Argentina Czech

CBS 316.90 Phoma medicaginisvar. medicaginis

GU238103 GU237828 GU237630 Medicago sativa Republic

CBS 560.81; PD 92/1569;PDDCC 6614

Phoma paspali T GU238124 FJ427048 FJ427158 Paspalum dilatatum New Zealand

CBS 561.81; PDDCC 6615 Phoma paspali GU238125 GU237889 GU237640 Lolium perenne New Zealand

CBS 249.92; PD 78/1088 Phoma subherbarum GU238144 GU237808 GU237658 Solanum sp. Peru

CBS 250.92; DAOM171914; PD 92/371

Phoma subherbarum B GU238145 GU237809 GU237659 Solanum sp. Peru

CBS 436.75 Phoma tropica T GU238149 GU237864 GU237663 Saintpaulia ionantha Germany

CBS 176.93; PD 86/547 Stagonosporopsisajacis

GU238167 GU237790 GU237672 Delphinium sp. Netherlands

CBS 177.93; PD 90/115 Stagonosporopsisajacis T

GU238168 GU237791 GU237673 Delphinium sp. Kenya

CBS 109171; PD 91/310;PDDCC 272

Stagonosporopsiscucurbitacearum

GU238180 GU237922 GU237685 Cucurbita sp. Netherlands

CBS 133.96; PD 79/127 Stagonosporopsiscucurbitacearum

GU238181 GU237780 GU237686 Cucurbita sp. New Zealand

CBS 425.90; PD 81/520 Stagonosporopsisinoxydabilis T

GU238188 GU237861 GU237693 Chrysanthemumparthenii

Netherlands

PD 85/259 Stagonosporopsisinoxydabilis

GU238189 GU237920 GU237694 Matricaria sp. Netherlands

CBS 500.63; MUCL 8090 Stagonosporopsischrysanthemi B

GU238190 GU237872 GU237695 Chrysanthemumindicum

Germany

CBS 137.96; PD 84/75 Stagonosporopsischrysanthemi B

GU238191 GU237783 GU237696 Chrysanthemumindicum

Netherlands

CBS 273.92; PD 76/1019 Stagonosporopsisvalerianellae

GU238200 GU237819 GU237705 Valerianellalocusta

Netherlands

CBS 329.67; PD 66/302 Stagonosporopsisvalerianellae B

GU238201 GU237832 GU237706 Valerianellalocustavar. oleracea

Netherlands

CBS 374.91; PD 78/391 Phoma eupyrena B GU238072 FJ426999 FJ427110 Solanumtuberosum

Netherlands

CBS 527.66; ATCC 22238 Phoma eupyrena B GU238073 FJ427000 FJ427111 Soil Germany

CBS 267.92; PD 76/1014 Phoma pereupyrena T GU238128 GU237814 GU237643 Coffea arabica India

Eur J Plant Pathol (2013) 137:127–136 131

329.67. The phylogenetic trees generated from the indi-vidual datasets produced nearly identical branching to-pologies (not shown). The combined dataset consisted of

2,163 characters including the alignment gaps (LSU:1327; ITS: 495 and TUB: 341). Of those characters,1884 (LSU: 1260; ITS: 403 and TUB: 221) were

Table 2 (continued)

Strain no.a Holomorphb GenBank accession numbers Host Origin

LSU ITS TUB

CBS 506.91; IMI 215229;PD 91/876

Phoma costarricensis B GU238058 GU237876 GU237596 Coffea sp. Nicaragua

CBS 320.90; PD 86/932 Stagonosporopsisdorenboschii B

GU238184 GU237830 GU237689 Physostegiavirginiana

Netherlands

CBS 426.90; IMI 386093;PD 86/551

Stagonosporopsisdorenboschii T

GU238185 GU237862 GU237690 Physostegiavirginiana

Netherlands

aATCC American type culture collection, Virginia, U.S.A., CBS Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands,CECT Colección Española de Cultivos Tipo, Valencia University, Spain, DAOM Canadian Collection of Fungal Cultures, Ottawa,Canada, IMI International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K., LEV Plant Health and DiagnosticStation, Auckland, New Zealand, MUCL Mycotheque de l’Universite catholique de Louvain, Louvain-la-Neuve, Belgium, PD PlantProtection Service, Wageningen, the Netherlands, PDDCC Plant Diseases Division Culture Collection, Auckland, New Zealandb T: Ex-type strain; B: Reference strain according to Boerema et al. (2004)

Fig. 1 The symptoms of blight of Adonis palaestina in the field. a,The earlier infected petioles above the ground surface; b, Thelesions covering most of the plant surface; c, The discrete yellow–

brown lesions on stems; d, The restricted brown stripe in the centralvascular tissue of longitudinal section of the root; e, The symptomsin bolting stage in field; f, The symptoms in late stage in field

132 Eur J Plant Pathol (2013) 137:127–136

constant, whereas 279 characters (LSU: 67; ITS: 92 andTUB: 120) were parsimony informative. Phylogeneticanalysis of the combined sequences showed that all P.adonidicola isolates from A. palaestina in China formeda unique and well-supported clade that grouped withthe S. ajacis reference strains (Fig. 4). These resultsindicates that the isolates of P. adonidicola from A.palaestina are conspecific, but different from otherStagonosporopsis or Phoma species.

Discussion

In this study, we have demonstrated that P. adonidicolawas the dominant colonist on the infected A. palaestina

plants and they caused typical brownish lesions oninoculated plants under artificially conditions in thegrowth chamber. Black–brown vascular tissues wereobserved only in some but not all of the diseased plantsin the field when the roots were longitudinally dissectedwhich was consistent with that observed in artificiallyinoculated plants. These findings thus suggested that P.adonidicola was the causal agent responsible for thediscolouration of the vascular tissue. In addition, theblack–brown vascular tissue of necrotic petiolesconnected to the stems was found on the plants, onwhich the black–brown vascular tissue of the rootswas also observed (Fig. 1d). These observations sug-gested that discolouring of the root vascular tissue in thefield probably resulted from stem infection. Our resultsthus show conclusively that stem blight, which is asso-ciated with significant losses of A. palaestina flowerproduction in Inner Mongolia since its first occurrencein 2004, is caused by the fungus P. adonidicola.

Previous studies showed that spots caused by P.exigua, which is closely related to P. adonidicola,occasionally occurred on Romaine lettuce leaves incontact with or close to the soil (Koike et al. 2006).In our study, the spot blight of A. palaestina caused byP. adonidicola was also first observed on plant partsnear the ground after rain events or irrigation. Thisis probably due to the high humidity near the soilwhich is beneficial to the pathogen. As with P.exigua, prolonged moist conditions are likely to benecessary for P. adonidicola to induce diseasesymptoms on A. palaestina.

Fig. 2 The frequency of isolating various fungi from diseasedAdonis palaestina plants. Phoma sp. (Pho); Plectosporium sp.(Ple); Alternaria sp. (Alt); Fusarium sp. (Fus); Colletotrichumsp. (Col). Values represent the means of 35 plants

Fig. 3 The symptoms on Adonis palaestina plants after inoculation with Phoma sp. or Plectosporium sp. a, Symptoms caused by Phomasp.; b, Pycnidia on the plant inoculated with Phoma sp.; c, Symptoms caused by Plectosporium sp

Eur J Plant Pathol (2013) 137:127–136 133

Here we show that Plectosporium sp. can also causespots on A. palaestina. However, the symptoms weredistinctly different from those caused by P. adonidicola(Fig. 3). Given the low incidence of recovery ofPlectosporium sp. from A. palaestina (Fig. 2), we donot believe that Plectosporium sp. is a primary pathogenin the blight disease on A. palaestina. The relationshipbetween Phoma and Plectosporium on A. palaestina is

not clear. Further studies should determine whetherthese fungi act synergistically to cause more diseaseseverity when present together.

The genus Phoma has been considered to be acommon and large fungal group (Montel et al. 1991),and it is usually difficult to identify the fungus to specieslevel. The identification system currently widely applieddivided the genus into nine sections, which is extremely

LY70

LY64

LY59

LY67

LY55

LY82

LY77

LY71

LY72

LY73

LY69

LY62CBS 176.93 Stagonosporopsis ajacis

CBS 177.93 Stagonosporopsis ajacis TCBS 273.92 Stagonosporopsis valerianellae

CBS 329.67 Stagonosporopsis valerianellae B

PD 85/259 Stagonosporopsis ligulicola var. inoxydabilis

CBS 425.90 Stagonosporopsis ligulicola var. inoxydabilis T

CBS 500.63 Stagonosporopsis ligulicola var. ligulicola B

CBS 137.96 Stagonosporopsis ligulicola var. ligulicola B

CBS 133.96 Stagonosporopsis cucurbitacearum

PD 91/310 Stagonosporopsis cucurbitacearum

CBS 320.90 Stagonosporopsis dorenboschii B

CBS 426.90 Stagonosporopsis dorenboschii T

CBS 506.91 Phoma costarricensis B

CBS 436.75 Phoma tropica T

CBS 101150 Boeremia exigua var. exigua

CBS 431.74 Boeremia exigua var. exigua B

CBS 267.92 Phoma pereupyrena T

CBS 123395 Phoma infossa T

CBS 123394 Phoma infossa

CBS 108.96 Phoma aquilegiicola B

CBS 107.96 Phoma aquilegiicola B

CBS 125.82 Epicoccum nigrum

CBS 173.73 Epicoccum nigrum T

CBS 527.66 Phoma eupyrena B

CBS 374.91 Phoma eupyrena B

CBS 250.92 Phoma subherbarum

CBS 249.92 Phoma subherbarum B

CBS 229.79 Phoma digitalis B

CBS 109179 Phoma digitalis

CBS 316.90 Phoma medicaginis var. medicaginis

CBS 544.74 Ascochyta hordei var. hordei

CBS 561.81 Phoma paspaliCBS 560.81 Phoma paspali T100

100

100

100

100

100

100

100

100

98

100

100

100

56

56

93

91

100

62

83

58

100

52

78

72

99

100

94

0.005

Fig. 4 Neighbour-joining treefor P. adonidicola and relatedspecies based on the com-bined sequence data for LSU,ITS, and TUB genes. Thepercentage numbers at thenodes indicate the levels ofbootstrap support based onneighbour-joining analyses of1,000 re-sampled data sets.The scale bar indicates 0.005substitutions per nucleotideposition. The tree was rootedwith Ascochyta hordei var.hordei (CBS 544.74) andPhoma paspali (CBS 561.81and 560.81). T: Ex-type strain;B: Reference strain accordingto Boerema et al. (2004)

134 Eur J Plant Pathol (2013) 137:127–136

helpful in morphological identification (Boerema et al.2004). However, this subdivision is primarily based on afew morphological or physiological characters and hasproved to be ambiguous and artificial (Aveskamp et al.2010). Aveskamp et al. (2010) reassessed the genericboundaries and rearranged the species and varieties inPhoma and related genera by combining morphologicaland molecular studies, which was thought to be morerealistic and reliable. P. adonidicola was morphologi-cally characterized in 2006 but not confirmed by mo-lecular biological studies. In this study, a phylogeneticanalysis was performed to elucidate its taxonomic posi-tion. All examined isolates of P. adonidicola were phy-logenetically close to Stagonosporopsis referencestrains, especially to S. ajacis, indicating that P. adoni-dicola and S. ajacis are close in genetic relationship.However, all examined isolates of P. adonidicola fromA. palaestina formed a unique and well-supported clade(Fig. 4) and P. adonidicola is morphologically differentfrom S. ajacis. The former species produced onlyone type of conidia (Li et al. 2006), while the latterspecies usually produced two types of conidia(Aveskamp et al. 2010). These results indicate thatthey are different species, even though they are closeto each other in phylogeny.

This study combined morphological data, sequencesof multiple genes, and pathogenicity tests to clarify thatthe primary causal agent of spot blight of A. palaestinain China is P. adonidicola. Such information would behelpful for growers to successfully manage the disease.Further research is required to gain information relatingto potential control measures and cultural practices forthe reduction of the disease.

Acknowledgments This research was financially supportedby grants from Program for Changjiang Scholars and InnovativeResearch Team in University (Grant number: IRT1042) and theprogram from Tongliao Academy of Agricultural Science inInner Mongolia. We are grateful to Dr. Xingzhong Liu of theInstitute of Microbiology, Chinese Academy of Sciences, andDr. Ching-Hong Yang of University of Wisconsin-Milwaukee,USA for critical discussions and reading of the manuscript.

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