2014 fungal diversity

Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China

Chao Xu&Chunsheng Wang&Liangliang Ju&Rong Zhang&

Alan R.Biggs&Eiji Tanaka&Bingzhi Li&Guangyu Sun

Received:6March2014/Accepted:2September2014

#School of Science2014

Abstract Apple ring rot inflicts severe economic losses in the main apple producing areas of East Asia.The causal agent of the disease has been variously identified as Macrophoma kuwatsukai,Physalospora piricola and Botryosphaeria berengeriana f.sp.piricola,although B.dothidea is currently the most widely accepted pathogen name.The taxonomic uncertainty has delayed research that is needed to manage effectively this destructive disease.In the present study,gene-alogical concordance phylogenetic species recognition (GCPSR)was applied to pathogenic fungal isolates from apple and pear from several locations in China,along with several reference isolates.Phylogenetic results based on se-quences of four nuclear loci(ITS,EF-1α,HIS and HSP) revealed the existence of two species within the examined isolates.One includes an ex-epitype isolate of B.dothidea and the other includes an isolate that was previously designated as B.berengeriana f.sp.piricola.Morphologically,the latter taxon presented an appressed mycelial mat on PDA whereas B.dothidea displayed columns of aerial mycelia reaching the lids,and conidia of the latter species were longer than B.dothidea.Botryosphaeria dothidea had a faster growth rate than the latter taxon under relatively high temperatures.Path-ogenicity tests showed that on pear stems the latter taxon caused large-scale cankers along with blisters whereas B.dothidea was non-pathogenic,but on apple shoots the two fungi induced large and small wart-like prominences, respectively.Overall,this cryptic species demonstrated suffi-cient genetic variations and biological differences from B.dothidea.As a result of taxonomic study,we described here the latter taxon in a new combination,Botryosphaeria kuwatsukai and designate an epitype.Both B.kuwatsukai and B.dothidea are considered to be the main causal agents for apple ring rot in China and Japan.

Key words Botryosphaeriaceae.Pear.Multi-gene phylogeny.Pathogenicity.Group I intron.Taxonomy Introduction

Ring rot has recently become one of the most destructive apple diseases in China,as well as in several neighboring countries,including Japan and South Korea(Ogata et al. 2000;Park2005;Tang et al.2012).Symptoms of the disease appear as a soft,light-coloured rot on fruit,especially during storage,and extensive cankers and/or warts on branches and trunks(Chen1999).Widespread planting of susceptible cul-tivars(e.g.,Fuji)over the past several years and reduced fungicide usage due to fruit bagging have likely resulted in the increased occurrence of ring rot,resulting in serious eco-nomic losses to Chinese apple growers(Kang et al.2009).

Apple ring rot disease was first reported in Japan in1907. The pathogen was later described as Macrophoma kuwatsukai Hara(Hara1930).Soon afterwards,the sexual morph of the pathogen was found and named Physalospora piricola(Nose 1933).This taxon had long been known as the causal agent

of C.Xu

:C.Wang:L.Ju:R.Zhang:G.Sun(*)

Key Laboratory of Crop Stress Biology in Arid Areas,College of

Plant Protection,Northwest A&F University,No.3Taicheng Road,

Yangling712100,Shaanxi,China

e-mail:sgy@https://www.wendangku.net/doc/2916026237.html,

B.Li(*)

College of Horticulture,Northwest A&F University,No.3Taicheng

Road,Yangling712100,Shaanxi,China

e-mail:bzhli530530@https://www.wendangku.net/doc/2916026237.html,

A.R.Biggs

Kearneysville Tree Fruit Research and Education Center,West

Virginia University,P.O.Box609,Kearneysville,WV25443,USA

E.Tanaka

Division of Environmental Science,Ishikawa Prefectural University,

Suematsu1-308,Nonoichi,Ishikawa921-8836,Japan

Fungal Diversity

DOI10.1007/s13225-014-0306-5

apple ring rot in other countries.Koganezawa and Sakuma (1980,1984)reappraised the sexual morph and tried to equate it with the morphologically identical Botryosphaeria berengeriana,a fungus causing apple Botryosphaeria canker (and fruit rot)in Japan.However,distinctly different symp-toms,cankers and wart bark,caused by B.berengeriana and by P.piricola,respectively,on branches and trunks resulted in the authors proposing the name Botryosphaeria berengeriana f.sp.piricola for Physalospora piricola(Koganezawa and Sakuma1984).As Botryosphaeria berengeriana and B.berengeriana f.sp.piricola induced the same apple rot symptom,diseases caused on fruit were referred to both as apple ring rot(Koganezawa and Sakuma1984).These two pathogen taxons are generally accepted in Japan,but they are rejected by European and American researchers who consider B.berengeriana to be a synonym of B.dothidea(Jones and Aldwinckle1990;Slippers et al.2004a).In China,the correct taxonomy of the apple ring rot pathogen is uncertain,in that Physalospora piricola,Botryosphaeria berengeriana, B.berengeriana f.sp.piricola and B.dothidea have been adopted by different researchers(Qu et al.2007;Peng et al. 2011;Lv et al.2012;Tang et al.2012).

The application of molecular techniques to the taxa asso-ciated with apple ring rot has shown that genetic heterogeneity exists among these pathogenic fungal isolates.Ogata et al. (2000),using ITS sequences,divided isolates of Botryosphaeria causing ring rot on apple fruit into two groups based on twig symptoms(warts or blight)and size of conidia. Huang and Liu(2001)showed that there was marked a dif-ference between B.berengeriana and B.berengeriana f.sp. piricola by RAPD analysis,in spite of their close genetic relationship.Peng et al.(2011)separated isolates from apple ring rot into two ISSR groups.Lv et al.(2012)proposed that the isolates causing apple ring rot with different variable sites in ITS sequences might behave differently in terms of patho-genicity and some biological characteristics(e.g.,the ability to sporulate).Xu et al.(2013)genotyped B.dothidea isolates (including some from apple trees)according to the distribution of ribosomal group I introns.Four genotypes were described and the authors indicated that there may be a correlation between these genotypes and host or geographic origin.The high amount of variation detected by the different approaches suggests that there is a mixture of pathogens rather than a single pathogenic species that causes apple ring rot.

Accurate identification of etiological factors is the founda-tion of plant disease research.Vague classification and con-fused appellation of the apple ring rot pathogen has led to the inability to compare research results from different countries, regions and investigators.Therefore,the objectives of this study were to:1)re-assess the Botryosphaeria isolates asso-ciated with symptoms of apple ring rot,including fruit rot, branch canker and wart bark in China,2)detect the reasons for the high amount of variation among isolates,and3)clarify whether any cryptic fungal species exist within the hypothet-ical pathogenic complex.Our approaches to the study includ-ed genealogical concordance phylogenetic species recognition (GCPSR)of multi-gene loci(Taylor et al.2000),as well as analysis of morphology,pathogenicity and growth characteristics.

Materials and methods

Fungal isolates

Twenty-four isolates(Table1)were used in this study,18of which were collected in the main apple production areas of China(Shaanxi,Henan,Shandong,Liaoning,Jiangsu,Shanxi, Hebei Provinces)during2008–2011.Of these18isolates,16 were either from warts or cankers on apple branches and trunks or apple fruit with rot symptoms;and two were isolated from the diseased woody tissue of pear exhibiting canker symptoms. The other six were reference isolates,including one isolate each of B.dothidea,B.berengeriana and B.berengeriana f.sp. piricola from the Fruit Tree Research Experiment Station of the Ministry of Agriculture,Forestry and Fisheries(MAFF), Japan,two isolates of B.dothidea from the International Col-lection of Microorganisms from Plants(ICMP),New Zealand, and one ex-epitype isolate of B.dothidea from the Centraalbureau voor Schimmelcultures(CBS),Netherlands. All the isolates collected in this research were obtained from either pycnidia directly or diseased tissue by cultivating on potato dextrose agar(PDA),and were examined for colony characteristics and microscopic morphology to preliminarily identify them as Botryosphaeria spp.They were then purified by single conidium isolation and stored at?80°C in College of Plant Protection,Northwest A&F University,China.

DNA extraction,PCR amplification and sequencing

Single-conidial isolates were grown on PDA and incubated for5days at25°C in the dark.Total genomic DNA was extracted from fungal mycelium following the modified phe-nol:chloroform DNA extraction method(Smith et al.2001). Four different gene regions were selected for characterization, including the complete nuclear rDNA internal transcribed spacer(ITS)region(White et al.1990),partial sequence of translation elongation factor1alfa(EF-1α),histone H3(HIS) and heat shock protein(HSP)genes(Inderbitzin et al.2010). The polymerase chain reaction(PCR)was performed in a reaction mixture of25μl containing approximately10–30ng fungal genomic DNA,10×Taq buffer with(NH4)2SO4, 1.5mM MgCl2,0.2μM of each dNTP,5pmol of each primer, 1U Taq polymerase and sterile ultrapure water.The following thermal protocol for PCR was applied:an initial denaturation at94°C for2min,followed by32amplification cycles of

Fungal Diversity

T a b l e 1C o l l e c t i o n d e t a i l s a n d G e n B a n k n u m b e r s o f s p e c i e s t r e a t e d i n t h e p h y l o g e n i e s

S p e c i e s

C u l t u r e s

G e o g r a p h i c o r i g i n H o s t S u b s t r a t e G e n B a n k n u m b e r s a

I T S E F 1-αH S P H I S I s o l a t e s c o l l e c t e d i n t h i s s t u d y B o t r y o s p h a e r i a d o t h i d e a P G 20S h a a n x i ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433398K J 433420K J 433466K J 433442B o t r y o s p h a e r i a d o t h i d e a P G 45S h a a n x i ,C h i n a M a l u s d o m e s t i c a t r u n k K J 433399K J 433421K J 433467K J 433443B o t r y o s p h a e r i a d o t h i d e a P G 77

S h a a n x i ,C h i n a M a l u s d o m e s t i c a b r a n c h K J 433400K J 433422

K J 433468

K J 433444

B o t r y o s p h a e r i a d o t h i d e a P G 267H e n a n ,

C h i n a M a l u s d o m e s t i c a f r u i t K J 433401K J 433423K J 433469K J 433445B o t r y o s p h a e r i a d o t h i d e a P G 293S h a n d o n g ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433402K J 433424K J 433470K J 433446B o t r y o s p h a e r i a d o t h i d e a P G 320L i a o n i n g ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433403K J 433425K J 433471K J 433447B o t r y o s p h a e r i a d o t h i d e a P G 327H e b e i ,C h i n a M a l u s d o m e s t i c a b r a n c h K J 433404K J 433426K J 433472K J 433448B o t r y o s p h a e r i a d o t h i d e a P G 329J i a n g s u ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433405K J 433427K J 433473K J 433449B o t r y o s p h a e r i a d o t h i d e a P G 331S h a n x i ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433406K J 433428K J 433474K J 433450B o t r y o s p h a e r i a k u w a t s u k a i P G 2/C B S 135219S h a a n x i ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433388K J 433410K J 433456K J 433432B o t r y o s p h a e r i a k u w a t s u k a i P G 55S h a a n x i ,C h i n a M a l u s d o m e s t i c a b r a n c h K J 433389K J 433411K J 433457K J 433433B o t r y o s p h a e r i a k u w a t s u k a i P G 259H e n a n ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433390K J 433412K J 433458K J 433434B o t r y o s p h a e r i a k u w a t s u k a i P G 297S h a n d o n g ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433391K J 433413K J 433459K J 433435B o t r y o s p h a e r i a k u w a t s u k a i P G 328H e b e i ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433392K J 433414K J 433460K J 433436B o t r y o s p h a e r i a k u w a t s u k a i P G 330J i a n g s u ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433393K J 433415K J 433461K J 433437B o t r y o s p h a e r i a k u w a t s u k a i P G 332S h a n x i ,C h i n a M a l u s d o m e s t i c a f r u i t K J 433394K J 433416K J 433462K J 433438B o t r y o s p h a e r i a k u w a t s u k a i L S P 5S h a a n x i ,C h i n a P y r u s s p .b r a n c h K J 433395K J 433417K J 433463K J 433439B o t r y o s p h a e r i a k u w a t s u k a i L S P 20

S h a a n x i ,C h i n a P y r u s s p .t r u n k K J 433396K J 433418

K J 433464

K J 433440

R e f e r e n c e s t r a i n s B o t r y o s p h a e r i a b e r e n g e r i a n a M A F F 645001

J a p a n M a l u s d o m e s t i c a

t w i g

K J 433409

K J 433431

K J 433479

K J 433455

B o t r y o s p h a e r i a b e r e n g e r i a n a f .s p .p i r i c o l a

M A F F 645002J a p a n M a l u s d o m e s t i c a t w i g K J 433397K J 433419K J 433465K J 433441B o t r y o s p h a e r i a d o t h i d e a

I C M P 8019

N e w Z e a l a n d P o p u l u s n i g r a

t w i g

A Y 236950

A Y 236899

K J 433476

K J 433452

B o t r y o s p h a e r i a d o t h i d e a I

C M P 13957N e w Z e a l a n d M a l u s ×d o m e s t i c a f r u i t K J 433407K J 433429K J 433477K J 433453B o t r y o s p h a e r i a d o t h i d e a M A F F 410826J a p a n P r u n u s s p .u n k n o w n K J 433408K J 433430K J 433478K J 433454B o t r y o s p h a e r i a d o t h i d e a C B S 115476

S w i t z e r l a n d

P r u n u s s p .

u n k n o w n

A Y 236949

A Y 236898

K J 433475

K J 433451

S e q u e n c e s u s e d B o t r y o s p h a e r i a a g a v e s M F L U C C 11-0125T h a i l a n d A g a v e s p .u n k n o w n J X 646791J X 646856N /A N /A B o t r y o s p h a e r i a a g a v e s M F L U C C 10-0051T h a i l a n d A g a v e s p .u n k n o w n J X 646790J X 646855N /A N /A B o t r y o s p h a e r i a c o r t i c i s C B S 119047U S A V a c c i n i u m c o r y m b o s u m u n k n o w n D Q 299245E U 017539N /A N /A B o t r y o s p h a e r i a c o r t i c i s A T C C 22927U S A V a c c i n i u m s p .u n k n o w n D Q 299247E U 673291N /A N /A B o t r y o s p h a e r i a d o t h i d e a P D 313U S A M a l u s d o m e s t i c a f r u i t G U 251101G U 251233G U 251629G U 251497B o t r y o s p h a e r i a d o t h i d e a P D 314U S A M a l u s d o m e s t i c a f r u i t G U 251102G U 251234G U 251630G U 251498B o t r y o s p h a e r i a d o t h i d e a

C B S 110302P o r t u g a l

V i t i s v i n i f e r a

u n k n o w n

A Y 259092

A Y 573218

N /A

N /A

Fungal Diversity

T a b l e 1(c o n t i n u e d )

S p e c i e s

C u l t u r e s

G e o g r a p h i c o r i g i n H o s t S u b s t r a t e G e n B a n k n u m b e r s a

I T S

E F 1-αH S P H I S

B o t r y o s p h a e r i a f a b i c e r c i a n a

C B S 127193C h i n a E u c a l y p t u s s p .u n k n o w n H Q 332197H Q 332213N /A N /A B o t r y o s p h a e r i a f a b i c e r c i a n a C M W 27108C h i n a E u c a l y p t u s s p .u n k n o w n H Q 332200H Q 332216N /A N /A B o t r y o s p h a e r i a f u s i s p o r a M F L U C C 10-0098T h a i l a n d E n t a d a s p .u n k n o w n J X 646789J X 646854N /A N /A B o t r y o s p h a e r i a f u s i s p o r a M F L U C C 11-0507T h a i l a n d C a r y o t a s p .u n k n o w n J X 646788J X 646853N /A N /A B o t r y o s p h a e r i a r a m o s a C B S 122069A u s t r a l i a E u c a l y p t u s c a m a l d u l e n s i s u n k n o w n E U 144055E U 144070N /A N /A B o t r y o s p h a e r i a s c h a r i f i i C B S 124703I r a n M a n g i f e r a i n d i c a u n k n o w n J Q 772020J Q 772057N /A N /A B o t r y o s p h a e r i a s c h a r i f i i C B S 124702I r a n M a n g i f e r a i n d i c a u n k n o w n J Q 772019J Q 772056N /A N /A C o p h i n f o r m a a t r o v i r e n s M F L U C C 11-0655T h a i l a n d E u c a l y p t u s s p .u n k n o w n J X 646801J X 646866N /A N /A C o p h i n f o r m a a t r o v i r e n s

C B S 117444

V e n e z u e l a E u c a l y p t u s u r o p h y l l a u n k n o w n K F 531822K F 531801N /A

N /A

C o p h i n f o r m a a t r o v i r e n s C B S 117450V e n e z u e l a E u c a l y p t u s u r o p h y l l a u n k n o w n E F 118051G U 134937N /A N /A

D o t h i o r e l l a i b e r i c a C B S 115041S p a i n Q u e r c u s i l e x t w i g A Y 573202A Y 573222G U 251696G U 251564D o t h i o r e l l a i b e r i c a C B S 113188S p a i n Q u e r c u s s u b e r u n k n o w n A Y 573198

E U 673278N /A N /A D o t h i o r e l l a s a r m e n t o r u m C B S 115038U K U l m u s s p .u n k n o w n A Y 573212A Y 573235N /A N /A M a c r o p h o m i n a p h a s e o l i n a C B S 227.33u n k n o w n Z e a m a y s u n k n o w n K

F 531825K F 531804N /A N /A M a c r o p h o m i n a p h a s e o l i n a P D 112U S A P r u n u s d u l c i s b a n d

G U 251105G U 251237G U 251633G U 251501N e o f u s i c o c c u m a n d i n u m C B S 117453V e n e z u e l a E u c a l y p t u s s p .u n k n o w n G U 251155G U 251287G U 251683G U 251551N e o f u s i c o c c u m l u t e u m C B S 110299P o r t u g a l V i t i s v i n i f e r a c a n e G U 251221G U 251353G U 251749G U 251617N e o f u s i c o c c u m m e d i t e r r a n e u m C B S 121558I t a l y O l e a e u r o p e a d r u p e G U 251175G U 251307G U 251703G U 251571N e o f u s i c o c c u m p a r v u m C M W 9081N e w Z e a l a n d P o p u l u s n i g r a u n k n o w n A Y 236943A Y 236888N /A N /A N e o f u s i c o c c u m p a r v u m C M W 10123S o u t h A f r i c a E u c a l y p t u s s m i t h i i u n k n o w n G U 251123G U 251255G U 251651G U 251519N e o f u s i c o c c u m r i b i s C B S 115475U S A R i b e s s p .u n k n o w n A Y 236935A Y 236877N /A N /A N e o f u s i c o c c u m r i b i s W A C 12395A u s t r a l i a E u c a l y p t u s p e l l i t a s t e m G U 251127G U 251259G U 251655G U 251523N e o f u s i c o c c u m v i t i f u s i f o r m e C B S 110887S o u t h A f r i c a V i t i s v i n i f e r a u n k n o w n A Y 343383A Y 343343N /A N /A N e o f u s i c o c c u m v i t i f u s i f o r m e W A C 12401A u s t r a l i a E u c a l y p t u s p a u c i f l o r a l e a f G U 251173

G U 251305

G U 251701

G U 251569

N e o s c y t a l i d i u m h y a l i n u m

C B S 145.78

U K H o m o s a p i e n s u n k n o w n K F 531816K F 531795N /A N /A N e o s c y t a l i d i u m h y a l i n u m P D 103U S A F i c u s c a r i c a l i m b G U 251106G U 251238G U 251634G U 251502N e o s c y t a l i d i u m h y a l i n u m

C B S 499.66

u n k n o w n M a n g i f e r a i n d i c a

u n k n o w n

K F 531820

K F 531798

N /A

N /A

N e o s c y t a l i d i u m n o v a e h o l l a n d i a e C B S 122071A u s t r a l i a C r o t a l a r i a m e d i c a g i n e a u n k n o w n E F 585540E F 585580N /A N /A N e o s c y t a l i d i u m n o v a e h o l l a n d i a e C B S 122610A u s t r a l i a A c a c i a s y n c h r o n i c i a u n k n o w n E F 585536E F 585578N /A N /A L a s i o d i p l o d i a t h e o b r o m a e P D 161U S A P i s t a c i a v e r a b r a n c h G U 251122G U 251254G U 251650G U 251518S p e n c e r m a r t i n s i a v i t i c o l a

C B S 117009S p a i n

V i t i s v i n i f e r a

c a n e

G U 251166

G U 251298

G U 251694

G U 251562

a

S e q u e n c e s g e n e r a t e d i n t h i s s t u d y a r e i n b o l d .T h e o t h e r s e q u e n c e s w e r e d o w n l o a d e d f r o m N C B I G e n B a n k r e f e r r i n g t o t h e w o r k o f I n d e r b i t z i n e t a l .(2010)a n d P h i l l i p s e t a l .(2013)

Fungal Diversity

denaturation at94°C for35s,annealing at their respective dependent temperatures for30s,elongating at72°C for3min and then one final step at72°C for10min.All PCR products (5μl)were electrophoresed on1%agarose gel and stained with the DNA dye,EZ-Vision One(Amresco,USA),and then visualized under UV illumination.PCR products were puri-fied and sequenced using the same forward and reverse primers at Sangon Biotech(Shanghai,China).

Sequence alignment and phylogenetic analysis

Raw sequences of isolates collected in this study and some reference isolates were obtained from ABI3730XL DNA Analyzer(Applied Biosystems,USA)and all the other se-quences used here were downloaded from GenBank(Table1) following Blast searches or references to published papers (Liu et al.2012;Hyde et al.2014).All nucleotide sequences were initially aligned with the software Clustal X2.0,and then imported into BioEdit5.0.9.1for optimizing manually to analyze their nucleotide polymorphisms(Hall1999;Larkin et al.2007).Phylogenetic reconstructions of concatenated and individual gene-trees were performed using Maximum-parsimony(MP)and Bayesian Markov Chain Monte Carlo criteria.In addition,to determine whether the combined se-quences can be used to construct phylogenetic analyses,sta-tistical congruence was examined by applying a partition homogeneity test(PHT)(Farris et al.1994).The PHT was performed in PAUP version4.0b10using1,000replicates and the heuristic standard search options(Swofford2003).

PAUP4.0b10was used to separately analyze single genes and combined genes to construct parsimony trees(Swofford 2003).Gaps were treated as“missing”and all characters were unordered and of equal weight.Insertions/deletions(indels), irrespective of their size,were each treated as one evolutionary event and weighted as one base substitution.MP trees were found using the heuristic search function with1,000random addition replicates and tree bisection and reconstruction(TBR) selected as branch swapping algorithm.Branches of zero length were collapsed and all multiple,equally parsimonious trees were saved.Branch supports were estimated using1,000boot-strap replicates(Felsenstein1985).Tree length(TL),consisten-cy index(CI),retention index(RI),rescaled consistency index (RC)and homoplasy index(HI)were calculated.

Bayesian inferences were performed using MrBayes3.1.2 for single locus datasets and for the combined dataset of multiple loci(Altekar et al.2004).The optimal nucleotide substitution models for each gene region and for the combined data were estimated using MrModeltest v2.2software (Nylander2004).The option for rates was set to invgamma, whereas all the other parameters of the likelihood model were default.Four simultaneous Markov chains were run starting from a random tree for5,000,000generations and trees were sampled every1,000generations.The first1,250of the5,000saved trees were discarded as burn-in,and the consensus tree was based on the remaining3,750trees.To determine the confidence of the tree topologies,values of Bayesian posterior probabilities(BPPs)were estimated using MrBayes.

Novel sequence data are deposited in GenBank(Table1) and the alignment in TreeBASE(https://www.wendangku.net/doc/2916026237.html,/phylo/ treebase/phylows/study/TB2:S15479).

Morphological analysis

Colony traits of single-conidium isolates on potato dextrose agar(PDA)medium were noted after a5-day incubation at 25°C in the dark.To induce sporulation,PDA dishes or malt extract agar(MEA)dishes filled with mycelia were incubated at25°C under alternating12h cycles of light/dark using both fluorescent and UV light for15days.In addition to the18 isolates collected from China’s apple-growing regions that were used for our phylogenetic analyses,18additional sam-ples that were collected similarly were used to make conidial measurements(36total isolates).Conidial measurements were made using conidia collected from pycnidia on the agar sur-face.Conidia were observed and measured from images taken using an Olympus BX51microscope with an Olympus DP72 digital camera(Olympus Corp.,Japan).Thirty measurements of conidial lengths and widths were taken for each isolate,and the ranges and averages as well as length and width ratio were calculated.SPSS statistical software was used to analyze variability in conidial lengths and widths among the isolates.

Growth rates testing

To assess colony growth rate,mycelial plugs(5mm in diam) of all test isolates were taken from the edge of3-day-old colonies and reversely transferred to the centers of9cm PDA dishes.Three replicates for each isolate were used. Colony diameters of each isolate were measured after5days of incubation at25,35and37°C in the dark,and their average growth rates were calculated and expressed as colony growth rate per24h.This experiment was conducted twice.Analysis of variance(ANOV A)of mycelial growth rate was performed using the ANOV A procedure of SPSS statistical software.

Pathogenicity testing

The pathogenicity of tested isolates was evaluated on shoots or stems of apple(Malus×domestica‘Fuji’)and pear(Pyrus pyrifolia Nakai‘Suli’).The pathogenicity test was conducted in an orchard(in Yangling,Shaanxi Province)from May to August,2011,and was repeated in2012.Scions were grafted on Malus micromalus,and then cultivated in the field with1.5-m×4-m spacing.Test isolates were grown on PDA medium at 25°C in the dark for3days before inoculation.The inoculation was performed on the non-wounded bark surface of2-year-old

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shoots of4-year-old apple saplings(3to3.5m high and15to 20branches)and2-year-old pear(1to1.5m high)stems.In mid-May,mycelial plugs(5mm in diameter)for each isolate were transferred to the inoculation positions that had been previously disinfected with70%ethyl alcohol,and then cov-ered with sterile moist cotton balls and wrapped with Parafilm to maintain high humidity.Five inoculations(10cm away from each other)were made on a tree as one replicate,and three replicates were used for each isolate;controls were treated in a similar manner with noncolonized PDA plugs.The inoculated trees were arranged in a randomized block design.In mid-August of those two years,the symptoms and sizes of lesions at the inoculated positions were examined,measured and re-corded.Measurements on the same tree were averaged,and then data among treatments in each repeated test were com-pared using ANOV A in SPSS statistical software.

Pathogenicity was tested also on detached non-wounded apple fruit in the laboratory.Mature apple(Fuji)fruit were washed,surface disinfested with3.5%NaOCl for5min, rinsed twice with sterile-distilled water,and dried in a transfer hood.Three mycelial plugs(5mm in diameter)cut from the margin of a5-day-old culture were placed on the surface of each non-wounded fruit with an equal distance between adja-cent plugs.Three fruits(replicates)were used for each isolate. Fruit inoculated with PDA plugs without fungus were used as controls.The incidence of lesions at the inoculated positions was determined on the second day after inoculation.

Re-isolation of fungi from diseased parts of inoculated shoots,stems and fruit was performed to complete Koch’s postulates.Warts or edges of the lesions were cleansed with 3.5%NaOCl for surface disinfestation,washed in sterile-distilled water twice,and then kept on PDA media at25°C for5days.Fungi were identified based on morphology and the sequence of the rDNA ITS region as described previously.

Intron analysis

The rDNA small-subunit(SSU)regions of all tested isolates were amplified and then sequenced to determine the presence or absence,distribution and primary structure of group I introns with reference to the method of Xu et al.(2013). Sequences of these introns were submitted to GenBank for BLASTN to clarify their attributes.

Results

Phylogenetic placement of tested isolates based on combined ITS and EF1-αsequences

A total of36sequences including four randomly selected tested isolates were comprised by the alignment of ITS and EF1-αregions,which contained760characters.In the parsi-mony analysis,555characters were constant,189characters were parsimony informative while16variable characters were parsimony uninformative.The first one(TL=342,CI= 0.7719,RI=0.9242,RC=0.7134,HI=0.2281)of100equally parsimonious trees yielded is presented here(Fig.1).The Bayesian tree(GTR model)agreed with the topology of the parsimonious tree,so Bayesian posterior probabilities are shown next to bootstrap values at the nodes.This phylogram provided a preliminary guide for identification of the isolates used in the research.In spite of low bootstrap values,isolates PG20and PG45cluster together and are evolutionarily rela-tively far apart from the clade that contains the isolates PG2 and LSP5.In terms of the topology of the tree,PG20and PG45are close to B.dothidea,whereas PG2and LSP5seem-ingly belong to an unrecognized taxon under the genus Botryosphaeria.

Phylogeny of individual and combined genes

ITS,EF-1α,HIS and HSP were used to construct phylogeny trees separately based on all18tested isolates and eight reference sequences.MrModeltest v2.2computed appropriate evolutionary models for each of the four loci used in Bayesian analyses as follows:GTR model for ITS,EF-1αand HSP,and HKY model for HIS.For all these genes(except ITS),topol-ogies of their trees are basically identical in the maximum parsimony and Bayesian inference.Only unrooted trees de-rived from the parsimony analysis are presented with the parsimony bootstrap values and posterior probabilities shown at the branches(Fig.2).Statistical data for individual trees are summarised in Table2.In the four gene genealogies,two distinct groups are consistently observed,of which nine tested isolates including PG20and PG45,B.berengeriana isolate MAFF645001and B.dothidea isolates PD314,CBS115476, ICMP13957,MAFF410826and ICMP8019belong to cluster A while the other nine tested isolates including PG2 and LSP5,B.dothidea isolate PD313and B.berengeriana f. sp.piricola isolate MAFF645002gather in cluster B.This partition is supported by high bootstrap values and posterior probabilities in EF-1α,HIS and HSP datasets,although slight variations still existed within cluster A according to the three genes(Fig.2b-d).For the ITS region,however,the two clusters are supported by low bootstrap values and they cannot be distinguished in Bayesian tree.Additionally,for sequences of genesβ-tubulin and Actin,too few parsimony-informative characters were found(Table2),so their phylogeny trees are not presented here.

The datasets including ITS,EF-1α,HIS and HSP were also analyzed collectively.Through the partition homogeneity test (Cunningham1997),no significant conflicts(P≥0.05)were found among them before catenation.The combined aligned data matrix contained37sequences including the outgroup

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Lasiodiplodia theobromae and 1610characters,of which 311characters were parsimony informative,185variable and parsimony uninformative,and 1114constant.The parsimony analysis resulted in a most parsimonious tree (TL=940,

CI=

Fig.1Phylogram inferred from combined parsimony analysis of ITS and EF1-αsequences from species of the genera Botryosphaeria ,Cophinforma ,Macrophomina ,Neoscytalidium ,Dothiorella and Neofusicoccum .The phylogenetic tree resulting from Bayesian inference had a topology identical to the MP tree presented.Bootstrap values/Bayesian posterior probabilities (>50%)are given at the nodes.The tree was rooted to Neofusicoccum luteum .Clades corresponding to genera and species are highlighted

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Fig.2Unrooted maximum-parsimony trees resulting from separate analysis of the sequences of ITS(a),EF-1α(b),HIS(c)and HSP(d).Bootstrap values/ Bayesian posterior probabilities(>50%)are indicated next to the branches.Two clusters(A and B)are respectively highlighted in dark and light green

Table2Information on the se-quence dataset and maximum parsimony(MP)trees for each locus

Locus

ITS EF-1αHIS HSPβ-tubulin Actin

Total no.of alignable characters502208506346373186 Total no.of variable characters1622531 No.of parsimony-informative characters1418501 No.of most parsimonious trees11002111 Tree length(TL)1622532 Consistency index(CI)111111 Homoplasy index(HI)000000 Retention index(RI)11110/01 Rescaled consistency index(RC)11110/01

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Fig.3Most parsimonious tree inferred from the combined four locus dataset of ITS,EF-1α,HIS and HSP.The phylogenetic tree resulting from Bayesian inference had a topology identical to the MP tree presented.Bootstrap values/Bayesian posterior probabilities(>50%)are given at the nodes.The tree was rooted to Lasiodiplodia theobromae.Clades corre-sponding to genera and species are highlighted

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0.6968,HI=0.3032,RI=0.8208,RC=0.5719)(Fig.3). Bayesian tree(GTR model)with an identical topology to the MP tree was reconstructed,and its posterior probabilities were thus added next to the bootstrap values.In the phylogenetic reconstruction of combined dataset,two deep clades were recognized and strongly supported with bootstrap values equal to100%and posterior probabilities of1.00,which completely corresponded to the two clusters formed in the individual gene genealogies.

Morphological characteristics

On PDA,colonies of all tested isolates were initially white, gradually becoming greenish brown to grey and then turning dark grey after5days of incubation.The reverse side of colonies was at first white,but after2or3days becoming dark green to olive green from the centre.No pigments were released to the medium by these pure cultures.For the isolates belonging to cluster A,bundles of aerial mycelia were com-monly observed and they could even reach the lids of Petri dishes.However,the isolates contained in cluster B generally produced compact and shorter aerial mycelia that can be described as an appressed mycelial mat(Fig.4a).

Conidial dimensions(lengths and widths)of17isolates belonging to cluster A and19isolates belonging to cluster B were measured in this study.Conidial lengths and widths of the cluster A isolates ranged from20.3to26.7μm and5.3to 7.0μm,respectively.Conidial lengths and widths of the cluster B isolates ranged from18.6to24.7μm and5.3to 7.2μm,respectively(Fig.4b).Average conidial length (23.3μm)of cluster A isolates was longer than that (21.9μm)of cluster B isolates(P<0.01),whereas there was no significant difference(P>0.01)in conidial width between these two groups of isolates.

Growth rates

Data from two repeated experiments on mycelial growth were not significantly different(P>0.05),and therefore were com-bined to calculate average colony diameter of each tested isolate.Isolates belonging to cluster A and cluster B were treated as two groups and there was no statistical difference (P>0.05)in growth rate among isolates of the same group when incubated at25,35and37°C,respectively.We then compared the average growth rates of these two groups of isolates at the above three incubation temperatures.At25°C, the average growth rates of cluster A and cluster B isolates were respectively12.4and11.9mm per24h and there was no difference(P>0.05)between them.When incubated at35°C, however,the growth rate(8.5mm per24h)of cluster A isolates was significantly faster than that(1.7mm per24h) of cluster B isolates(P<0.01).At37°C,no visible colony development was observed for any isolates of cluster B,whereas cluster A isolates could still extend slowly at an average growth rate of3.3mm per24h(Fig.5). Pathogenicity

Rounded protuberances were commonly visible approximate-ly40to60days after inoculating apple shoots with mycelial plugs.They presented distinctly different sizes and denseness between cluster A and cluster B isolates(P<0.01),and here only measurement data of seven inoculation treatments(six isolates plus one control)are presented(Table3).Smaller protuberances(0.7–1.0mm in diameter)induced by the clus-ter A isolates were distributed densely,and described as a verruculose surface,whereas the cluster B isolates produced bigger but sparser protuberances(3.0–4.1mm in diameter), described as a verrucose symptom,which occasionally broke through the epidermis(Fig.6).As the infection progressed, the protuberances usually increased in number,darkened in colour and eventually cracked.When these isolates were inoculated on pear stems,more complex symptoms were observed after about6weeks(Table3,Fig.6).For the cluster A isolates,just small areas around the inoculation positions turned brown(i.e.,local necrosis).In this situation,the above isolates could be considered non-pathogenic on pear trees.For the cluster B isolates,however,the symptoms were extensive and necrosis extended on a large scale into the phloem tissues (cankers15.0–17.5mm in diameter).Additionally,blisters (2.5–3.0mm in diameter)were noticed to form over the cankers and occasionally cause splitting of the periderm in the necrotic areas.No symptoms were observed on apple and pear trees that belonged to the control group

On apple fruit,all tested isolates caused brown soft rot symptoms on non-wounded fruit,and exudation could be observed in the lesion areas.There was no marked difference between cluster A and cluster B isolates in pathogenicity.Fruit that were inoculated with PDA plugs without fungi remained healthy.

Analysis of introns

The rDNA SSU of18tested isolates used in this study(not published)were scanned for group I introns that related to the genetic diversity(four genotypes)within B.dothidea discussed in our previous research(Xu et al.2013).As a consequence,all cluster A isolates possessed a1349-bp intron (Bdo.S1199-A)at position1199,which agreed with the ge-notype I of B.dothidea.The cluster B isolates were detected to have the same primary nucleotide structure of rDNA SSU as genotype II of B.dothidea that was characterized by two group I introns(Bdo.S943and Bdo.S1506)inserted at posi-tions943and1506,respectively(Fig.7).

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Taxonomy

Based on these individual gene genealogies (ITS,EF-1α,HIS and HSP),multi-locus phylogeny inferred by their combined alignment and biological characteristics,the 18tested isolates associated with branch or fruit ring rot of Malus×domestica

and Pyrus pyrifolia were shown to belong to cluster A and cluster B,respectively,which should be identified as two species of Botryosphaeria ,i.e.,a previously described species B.dothidea and one new taxon.

Botryosphaeria kuwatsukai (Hara)G.Y .Sun and E.Tana-ka,comb.nov.et.emend (Fig.8)MycoBank:MB 808073.

Facesoffungi Number:FoF00170.

Basionym:Macrophoma kuwatsukai Hara,Pathologia Agriculturalis Plantarum 482(1930)

=Physalospora pyricola Nose,Ann https://www.wendangku.net/doc/2916026237.html,.-Gen.Chosen 7.161(1933).(as “piricola ”)

≡Guignardia pyricola (Nose)W.Yamamoto,Sci.Rep.Hyogo Univer.Agric.5.11(1961).(as “piricola ”)

=Botryosphaeria berengeriana De Notaris f.sp.pyricola Koganezawa &Sakuma,Bull.Fruit Tree Res.Stat.,C11:58,1984.(as “piricola ”)

Lectotype designated here (MB 808072):illustration of Macrophoma kuwatsukai Hara,Pathologia Agriculturalis Plantarum 482:figure 191(1930

)

Fig.4 a.The colony

morphology of cluster A isolates (left)and cluster B isolates (right)from above after 5-day cultivation on PDA at 25°C.b.The average lengths and widths of 30conidia measured for each of 36tested isolates.Seventeen cluster A isolates and 19cluster B isolates are indicated on the graph as blue triangles and red squares,

respectively

Fig.5Mean mycelium growth rates (mm per 24h)obtained for isolates of cluster A and cluster B 5days after inoculation on PDA at 25,35and 37°C.Bars above columns are the standard error (5%)of the mean

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Epitype:CHINA,Shaanxi Province,Yangling,from fruit of M.×domestica,7September2011,CS Wang,dried culture, HMAS245112(PG2);living cultures derived from ex-epitype—CBS135219=CGMCC3.15244.

Colonies on PDA attaining52mm diam.after4days at25°C in the dark,initially white with moderately dense,appressed mycelial mat and aerial mycelium without columns,gradually becoming grey to dark grey.The reverse side of the colonies at

Table3Symptoms on apple shoots and pear stems inoculated with isolates of cluster A and cluster B via the mycelium plug method

Apple Pear

Isolate Symptom Size(mm)Symptom Canker size(mm)Blister size(mm)

Cluster A

PG77Verruculose0.7–0.9–––

PG320Verruculose0.9–1.0–––

PG329Verruculose0.8–0.9–––

Cluster B

PG2Verrucose 4.0–4.1Canker with blister16.7–17.5 2.7–2.8

PG259Verrucose 3.0–3.6Canker with blister16.0–16.3 2.6–3.0

PG330Verrucose 3.5–3.8Canker with blister15.0–15.5 2.5–2.8 Control––––

–

Fig.6Symptoms on non-

wounded apple and pear shoots or

stems one or two months after

inoculation(via mycelial plugs)

with different isolates from cluster

A and cluster B.a.Small

protuberances on apple shoots.b.

Large protuberances on apple

shoots.c.Localized necrotic spots

on pear stems.d.Cankers with

blisters on pear stems.a-1and

c-1:PG45.b-1and d-1:PG2.

a-2and c-2:PG293.b-2and d-2:

PG330.a-3and c-3:PG327.b-3

and d-3:LSP20.4:Control

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first white,but after 2–3days becoming dark green to olive-green from the centre.This colouration gradually spreads to the edge and becomes darker from the centre until the entire underside of the colony is black.Conidiomata absent in culture on PDA or on MEA in the dark,formed under the 12-h interactive treatment with black light and fluorescent lamp within 15–20d,superficial,dark brown to black,globose,mostly solitary and covered by mycelium.Conidiogenous cells holoblastic,hyaline,sub-cylin-drical,7–18×2–4μm,proliferating percurrently with one or more proliferations and occasionally resulting in periclinical thickening.Conidia produced in culture similar to those formed in nature,narrowly fusiform,or irregularly fusiform,base subtruncate to bluntly rounded,smooth with granular contents,widest in the middle to upper third,(18.5–)20–24.5(?26)×5–7(?8)μm (mean±SD=22.3±2.1×6.2±0.9μm,n=60,L/W ra-tio =3.6),forming 1–3septa before germination.Microconidiomata globose,dark brown to black.Microconidiophores hyaline,cylindrical to sub-cylindrical,3–10×1–2μm.Microconidia unicellular,hyaline,allantoid to rod-shaped,3–8×1–2μm.Sexual state not observed in culture.Known hosts:Malus×domestica and Pyrus pyrifolia Known distribution:China,Japan and North America Additional material examined:CHINA,Shaanxi Province,on branch of M.×domestica ,2011,CS Wang,culture PG 55;Henan Province,on fruit of M.×domestica ,2011,CS Wang,culture PG 259;Shandong Province,on fruit of M.×domestica ,2011,CS Wang,culture PG 297;Hebei Province,on fruit of M.×domestica ,ZQ Zhou,culture PG 328;Jiangsu Province,on fruit of M.×domestica ,ZQ Zhou,culture PG 330;Shanxi Province,on fruit of M.×domestica ,ZQ Zhou,culture PG 332;Shaanxi Province,on branch of Pyrus pyrifolia ,2012,CS Wang culture LSP 5;Shaanxi Province,on fruit of P .pyrifolia ,2012,CS Wang,culture LSP 20;JAPAN,Iwate,on twigs of M.pumila Mill.var.domestica ,1980,H.Koganezawa,culture MAFF 645002.

Notes :Miura (1917)found ring rot disease on pears and named the fungus as “Coniothecium ”.Kuwatsuka (1921)said that the fungus belonged to “Macrophoma ”,and Hara (1930)described it as Macrophoma kuwatsukai Hara.The original description was only in Japanese.Nose (1933)found the sexual stage,which he described as Physalospora piricola Nose,also with only a Japanese description.Yamamoto (1961)transferred the fungus to Guignardia piricola (Nose)W.Yamamoto,but this was not accepted by others.Koganezawa and Sakuma (1984)proposed the name,Botryosphaeria berengeriana De Notaris f.sp.piricola Koganezawa &Sakuma for the fungus causing apple wart bark to separate typical B.berengeriana (=B.dothidea )caus-ing cankers on apple and pear.Even though there was no Latin description or diagnosis for both Macrophoma kuwatsukai and Physalospora piricola ,they are validly published (Art.39.1).Macrophoma kuwatsukai has priority over Physalospora piricola ,even though it is an asexual stage name (Art.59.1),and should be treated as the basionym.Botryosphaeria kuwatsukai ,which had been always confounded with B.dothidea ,was described and identi-fied in this study based on multiple locus genealogies.Biological characteristics including aerial mycelia growth,mycelial growth rate and pathogenicity also supported the segregation of these two species.Morpho-logically,however,it is hard to discriminate B.kuwatsukai from B.dothidea as they produce similar conidia.In the original description of Macrophoma kuwatsukai Hara (1930),the holotype was not designat-ed.The illustration should be treated as the lectotype.Based on the lectotype,it is hard to discriminate it from other morphological similar species,thus we designate an epitype.

Botryosphaeria dothidea (Moug.ex Fr.)Ces.&De Not.,Commentario della SocietàCrittogamologica Italiana 1(4):212(1863)

Material examined:CHINA,Shaanxi Province,on fruit of M.×domestica ,2011,CS Wang,culture PG 20;Shaanxi Province,on trunk of M.×domestica ,2011,CS Wang,culture PG 45;Shaanxi Province,on branch of M.×domestica ,2011,CS Wang,culture PG 77;Henan Province,on fruit of M.×domestica ,2011,CS Wang,culture PG 267;Shandong Province,on fruit of M.×domestica ,2011,CS Wang,culture PG 293;Liaoning Province,on fruit of M.×domestica ,ZQ Zhou,culture PG 320;Hebei Province,on branch of M.×domestica ,ZQ Zhou,culture PG 327;Jiangsu Province,on fruit of M.×domestica ,ZQ Zhou,culture PG 329;Shanxi Province,on fruit of M.×domestica ,2012,CS Wang,culture PG 331;SWITZERLAND,Crocifisso,isolated from Prunus sp.,October 2000,B.Slippers,culture CBS 115476;NEW ZEALAND,Bay of Plenty,Te Puke,from bark of dead twig of

Populus

Fig.7Primary structures of the rDNA SSU in cluster A isolates and cluster B isolates.The entire SSU rDNA is shown by the blue rectangle.The triangles in purple,green and yellow indicate three different group I introns.The position,length and name of each intron are given separately above,in and below the triangles.The insertion sites correspond to the 16S rRNA of E.coli J01859

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nigra ,1December 1981,G.J.Samuels,culture ICMP 8019;NEW ZEALAND,Waikato,on fruit of M.×domestica ,1April 1999,M.A.Manning,culture ICMP 13957;JAPAN,IBARAKI,isolated from Prunus sp.,May 1993,T.Yamada,culture MAFF 410826;JAPAN,Iwate,on twigs of M.pumila Mill.var.domestica ,1978,H.Koganezawa,culture MAFF 645001.Notes :Botryosphaeria dothidea was isolated as the patho-gen causing apple ring rot from fruit and branches of Malus×domestica in this study.The colony characteristics and micro-scopic morphology is exactly similar to the ex-epitype of B.dothidea (Slippers et al.2004a ;Liu et al.2012;Hyde et al.2014).In the phylograms,our nine isolates confidently clustered together with the ex-epitype isolate of B.dothidea (CBS 115476)and other referential B.dothidea

isolates

Fig.8Asexual structures of Botryosphaeria kuwatsukai (from ex-epitype CBS 135219).a .Infected fruit of apple (black conidiomata scattered on the lesion)b .Culture on PDA (5days old)c.Conidiomata on PDA d .Conidia and microconidia e -g .Conidia before germination

with 1–3septa h .Microconidiomata i .Microconidiophores j .Conidia k -l .Conidiophores and conidiogenous cells.Scale bars:c=500μm d,f,h and i=10μm e,g=5μm i,j and l=20μm

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including ICMP8019,ICMP13957,MAFF410826and MAFF645001(Figs.2,and3).

Discussion

The theory of multiple gene genealogies has been increasingly applied in studies of species boundaries in both human and plant pathogenic fungi,revealing vast numbers of cryptic species and species complexes in fungal taxa previously iden-tified as one morphospecies(Pringle et al.2005;Hyde et al. 2010,2014;Liu et al.2012;Maharachchikumbura et al.2012; Udayanga et al.2012;Morgado et al.2013).In Botryosphaeriaceae,for example,Slippers et al.(2004a) redefined the circumscription of B.dothidea sensu stricto and separated out B.parva and B.ribis that had previously served as synonyms of B.dothidea.Pavlic et al.(2009)revealed three cryptic species within the Neofusicoccum parvum/N.ribis com-plex isolated from Syzygium cordatum trees in South Africa. Furthermore,Diplodia scrobiculata was described as a sister species of D.pinea(De Wet et al.2003)and N.eucalypticola and N.australe were perceived as sister species of N.eucalyptorum and N.luteum,respectively(Slippers et al. 2004b,c).In this study,Botryosphaeria kuwatsukai is described and identified from pathogenic isolates causing ring rot of apple and pear trees in China,thus proving our hypothesis that the previous ring rot pathogen,B.dothidea,is a species complex. This cryptic species was recognized primarily based on DNA sequence data of four nuclear genes combined with some phe-notypic characters as supplementary evidence.

According to the phylogram of combined ITS and EF1-αsequences(Fig.1),we can estimate that the four randomly selected tested isolates belong to two different taxa(one probably equals to B.dothidea)in Botryosphaeria.Further analysis of the genealogies of EF-1α,HIS and HSP genes and their combination(Figs.2,and3)demonstrated that all tested isolates were subsumed in two genetically well-separated clusters(cluster A and cluster B),which correspond to the above two taxa.In cluster A,despite some slight variations, nine tested isolates gathered well with five reference B.dothidea isolates including the ex-epitype CBS115476, indicating that they truly belong to B.dothidea.In cluster B, the remaining nine tested isolates and two reference B.dothidea isolates were re-identified and named as B.kuwatsukai,thus suggesting that previous B.dothidea iden-tified from apple ring rot was a species complex.Two other genes,β-tubulin and Actin,widely used in phylogenetics, were also analyzed here.They were more inclined to make all tested isolates fall together in the B.dothidea clade.This is consistent with the result of Tang et al.(2012),who considered according to the multi-gene sequence data of ITS,β-tubulin and Actin that isolates associated with symptoms of apple and pear ring rot(warts,cankers and fruit rot)all belong to the same pathogen,B.dothidea.

In microscopic morphology,B.kuwatsukai and B.dothidea exhibited a statistically significant difference in conidial length.However,this variation represented a continuum among isolates of the B.dothidea/B.kuwatsukai complex and,therefore,it was too difficult for us to use this character to set a clear delimitation of groups,and then to screen B.kuwatsukai out of B.dothidea prior to the application of some other effective methods.This indicates that genetically isolated species do not necessarily show divergence in some morphological characters such as conidial morphology,which is consistent with conclusions of several previous studies (Pavlic et al.2009;Maharachchikumbura et al.2012; Udayanga et al.2012;Muggia et al.2014).Colony morphol-ogy and different growth patterns of aerial mycelia(i.e., appressed mycelial mat from B.kuwatsukai and columns of aerial mycelia from B.dothidea)can be used to preliminarily distinguish these two species.

When cultured below their optimal temperatures(generally 22to28°C)in darkness,both B.kuwatsukai and B.dothidea grew faster on PDA as temperature increased and there was no significant difference between their growth rates(unpublished data).When cultured above their optimal temperatures,both species grew more slowly as temperature increased;however, the decline in growth rate was more pronounced with https://www.wendangku.net/doc/2916026237.html,pared to the demanding requirement for techniques and equipment used in molecular identification,it is undoubtedly a more expeditious approach to differentiate the two species by simultaneously cultivating them at rela-tively high temperatures(e.g.,35°C)and then assessing their colony diameters after3–5days.

Although the host affiliations of isolates collected in this study were limited to apple and pear,host ranges of the two fungi could be inferred according to previous research (Inderbitzin et al.2010;Marques et al.2013;Xu et al.2013). As mentioned above,the isolates of B.kuwatsukai actually correspond to the genotype II of previous B.dothidea,whereas the other three genotypes(III and IV were not isolated here)are still considered to be genetically different populations within current B.dothidea.Through investigation and statistics,Xu et al.(2013)found that genotype II isolates of B.dothidea could be isolated only from apple and pear,and was the only popu-lation detected on pear,whereas the other three genotypes of B.dothidea collectively infected dozens of shrubs and trees, except pear.From the above,it is conjectured that B.kuwatsukai may have host specificity for apple and pear.

The phylogenetic analyses showed that both species caus-ing apple ring rot belong to the genus Botryosphaeria(Liu et al.2012;Hyde et al.2014).The Japanese B.berengeriana isolate MAFF645001belonged to B.dothidea,which agrees with the conclusion of Slippers et al.(2004a),whereas another Japanese isolate B.berengeriana f.sp.piricola MAFF645002

Fungal Diversity

and B.dothidea isolate PD313from the USA were both re-identified as B.kuwatsukai,which suggests that this species is a cosmopolitan fungus,not unique to China.According to the morphological and biological description of B.berengeriana f.sp.piricola by Koganezawa and Sakuma(1984),this fungus is morphologically identical with B.dothidea,grows slower than B.dothidea and mainly causes ring rot of apple and pear. All these characters are shared by B.kuwatsukai,supporting our phylogenetic results.Reference isolates PD313and PD314(Inderbitzin et al.2010),which were isolated from fruit of M.domestica in USA,were once both described as B.dothidea and caused a prevalent disease called apple white rot(Jones and Aldwinckle1990;Inderbitzin et al.2010). However,in this study the two isolates were classified as different species,indicating that apple white rot,which is widely distributed in the North America,is probably able to be induced by two pathogens, B.dothidea and/or B.kuwatsukai.

Pathogenicity tests revealed that both B.kuwatsukai and B.dothidea could induce protuberances on the surfaces of apple shoots,with the two species associated with protuber-ances of different size and density(described as verrucose and verruculose,respectively).Protuberances caused by B.kuwatsukai were nearly four times as larger than those by B.dothidea.Previously,this phenomenon was often attributed to differences in virulence of different B.dothidea isolates and was applied to the establishment of evaluation criteria for host resistance(Zhou et al.2010;Lin et al.2011).Our results show that size of protuberances is not related to virulence differences and this character should not be applied when screening for resistance.

The common canker symptom of apple ring rot on shoots was not observed in the inoculation tests.Koganezawa and Sakuma(1984)inoculated mycelial plugs of B.berengeriana (B.dothidea)isolates and B.berengeriana f.sp.piricola (=B.kuwatsukai)isolates on wounded apple trunks,and re-ported the former produced typical cankers while the latter formed rough callus bark on inoculation sites.They also inoculated spore suspension of the two pathogens on non-wounded apple trunks,and consequently B.berengeriana caused no symptoms while B.berengeriana f.sp.piricola produced typical wart-like protrusions.Tang et al.(2012) performed similar pathogenicity tests with several B.dothidea isolates,in which warts formed only on non-wounded apple shoots whereas cankers often appeared on wounded apple shoots.Therefore,we speculate that different inoculation methods(non-wounded or wounded and mycelium plugs or spore suspension)lead to different symptoms on apple trunks.

B.kuwatsukai tends to infect hosts from the lenticels and causes wart-like protuberances,whereas B.dothidea tends to infect hosts from wounds and causes cankers and,in fact, when the inoculum dose is enough(e.g.,mycelium plugs), B.dothidea can cause similar symptoms as B.kuwatsukai (Koganezawa and Sakuma1984;Zhang et al.2011).On pear stems,large-scale cankers along with blisters were produced by B.kuwatsukai,whereas B.dothidea just induced localized necrotic spots,which could be regarded as non-infectious. This agrees with the view of Koganezawa and Sakuma (1984)that B.berengeriana f.sp.piricola(=B.kuwatsukai) is the true and only pathogen of pear ring rot.

Our research provided sufficient evidence to prove that apple ring rot disease is caused by two different pathogens, B.dothidea and B.kuwatsukai,whereas B.kuwatsukai alone is the pathogen responsible for pear ring rot.With increased understanding of the etiology of apple ring rot,we can begin to develop targeted management strategies based on sanita-tion,cultural methods,chemical methods and resistance breeding.

Acknowledgments We are grateful for help in sample collecting by Prof.Zengqiang Zhou(Zhengzhou Institute of Pomology,Henan,China) and Prof.Meng Zhang(Henan Agricultural University,Henan,China). We thank Prof Pedro W.Crous(CBS-KNAW Fungal Biodiversity Cen-tre,The Netherlands.)and Dr Eric H.C.McKenzie(Landcare Research, Auckland,New Zealand)for exchanging the authentic cultures and giving suggestion in nomenclature.This work was supported by National Natural Science Foundation of China(31371887,31171797),the111 Project from Education Ministry of China(B07049),Specialized Re-search Fund for the Doctoral Program of Higher Education (20130204110002)and China Agriculture Research System(CARS-28). References

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2014江苏省中小学教师心理健康网络知识竞赛(100分二)

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（二）抓好项目建设，破解制约供销社发展的瓶颈 1.积极推进现代化农资配送中心项目建设。市生产资料公 司作为我市最大的农资销售龙头企业，初步构建了覆盖全市的农 资流通网络，发挥了农资销售主渠道作用。但目前生产资料公司 经营场地狭小，仓储设施陈旧，道路狭窄，车辆通行困难，严重影响了农资配送业务的开展，因此，我们计划新建一座现代化农资配送中心。目前，我们已完成了农资配送中心项目的前期设计规划，正在积极寻找地源。 2 .抓好盐业公司升级改造项目。盐业公司经营设施老化， 房屋破旧，硬件设施不达标，阻碍了企业的发展，我们积极筹措资金，抓好盐业公司配送中心升级改造项目，目前，完成了对盐业公司仓库、办公住房的重建，对沿街门店进行了整体装修，着力打造AAA级和谐企业。 3.抓好土产公司外迁项目。土产公司承担着全市烟花爆竹的供应任务，该公司地处居民区，交通拥挤，出行困难，安全问题日益突出，市安监局已多次要求外迁，为此，我们计划征地30亩，做好外迁工作。目前，正在积极争取用地指标。 （三）抓好龙头企业建设，打造农村现代流通服务体系 1 .抓好农资销售龙头企业建设。我们以市丰田农资公司为 龙头，16个基层社，150家农资农家店，190个农资零售网点为销售终端，大力开展农资连锁经营业务，积极与中农、省农资公司和农资生产厂家合作，实行厂商联合，开展总代理总经销业务，

2012年度总结及2013年度计划

2012年度总结及2013年度计划D

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供了保障。充分利用新车站站场宽阔、停车方便、设施先进、服务周到等优势，吸引旅客到新车站购票乘车，聚集了客源，实现了主营业务稳步发展。客运站进站班车38台，日发班车50班次，上半年共发出班车8710班次，同比增长10.43%；输送旅客11.23万人次，同比增长10.48%；组织加班178班次，同比增长4.09%，包车38班次，同比减少15.8%。其中公司17台自营车发班3702班次，同比减少 %；运送旅客8.15万人次，同比减少3.54%。农村客运总站进站经营车辆21台，日发班车42班，上半年发出班车7699班次，同比增0.26%，输送旅客6.44万人次，同比增8.7%。 （二）全力以赴抓好重大节假日旅客运输工作，安全增收创效益。 2012年春运，我司精心组织，周密部署，积极组织客源和运力，增加加班、包车台次，尽量满足旅客要求，让旅客走得安全、及时、有序、满意，春节运输安全、和谐、高效。客运站和农村客运站春运共售票547.81万元，发出班车3538班次，输送旅客5.05万人次。其中客运站售票533万元，发出班车2026班次（加班包车176班），发送旅客3.57万人次，同比分别增长11.51%、10.22%和11.55%。公司投入14台自营车参加春运，营运收入399.28万元，发班898班次（加班34班），运送旅客2.21万人次，取得了良好的经济效益. “五·一”、端午等重大节假日，我司同样精心组织，合理安排，尽最大的人力、运力输送旅客，增加营运收入，为全年运输生产任务的完成打下基础。 （三）坚持公司、生产部门领导值班和公司生产会议制度。每天召开生产碰头会，每月召开司务会，及时了解掌握企业生产经营情况、生产经营中出现的问题、以及各项管理工作落实情况，提出完善和整改意见，协调部门工作，保证了生产任务、各项工作的完成。每月召开一次经济活动分析会，各部门对生产经营指标完成情况进行分析对比，查找存在的问题，

2014年流行色

2014年春夏秋冬流行色是什么时尚总是轮回的，每一次的循环与回归都预示着下一季潮流的卷土重来，2014年，最流行什么颜色是不少爱美人士关注的焦点，具体流行什么颜色，请看图文解说。 1.海洋蓝 海洋蓝将是2014年不退色的潮流，今年的海洋蓝，更偏向于金属的淡淡光泽感，这似乎是今年流行色的共同点，少了几分低调，凭添更多华贵的气质。 2.黄与红 这一系列是大地色系的变异，看着这2个颜色，是不是有浓浓的拉丁风情，席卷而来的感受呢，这是很热情、温暖的颜色，适合熟女穿，比较有亲和力。

3.白加黑 无需多说，黑白是经久不过时的颜色，无论在哪个年代，有这两种颜色撑场面，相比也不会逊色太多。感觉黑色鸭梨山大的，可以选择深灰色减轻负重感。

4.富贵金 2014年必定大热的一款颜色，模特儿们还勉强能撑起这么华美的颜色，像一般路人穿这种颜色，估计是hold不住的，一股浓浓的土豪即视感。

5.渐变紫 渐变是时尚的永恒主题，无论什么时候，渐变都会存在，不过个人不建议买渐变紫颜色的衣服，因为，东方人的皮肤在紫色的映衬下会更加显暗淡。

色彩机构潘通Pantone发布2014年度流行色蝴蝶兰紫 色彩专家Pantone公司最近发布了2014的年度流行色。你还记得2010年的流行色是绿松石，所以一整年我们都随处可见这种蓝绿色的身影，从明星们的眼影，到T台上模特们的指甲，再到新上市的彩妆盘。到了2011年，最热门的颜色变成了“忍冬花”（honeysuckle），这种淡淡的粉红色调能给肌肤带来健康的光泽，这种光泽被认为是魅力、活力和表现，能振奋人的精神。2012年，流行色是橘色，所以在T台我们总能看到饱和鲜艳的橘色唇膏带来的十足热力。今年的流行色则是Radiant Orchid，也就是蝴蝶兰紫。

江苏省中小学教师继续教育网络培训答案

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答案： (A)采访表 (二)多选题 1. 在第四步骤：编制班级方案的过程中，我们要遵循的原则是（）。 答案： (A)说服性 (B)实用性 (C)法律性 (D)全体性 2. 视频中，在第三步骤收集班级所要研究问题的有关资料时，我们可以通过实地查看、采访相关人员、上网、看电视、（）（）这些渠道来收集资料。 答案： (A)去新华书店 (B) 查看报刊、书籍 (C)去图书馆 (D)发放调查问卷 3. 在公民教育实践活动与学科教学整合的过程中，需要着力做好（）。 答案：(A)将公民课题纳入原有课程体系进行课程定位(B)确立新课程的学习目标(C)精心进行教学活动的设计与实施(D)重视学习成果展示和学习反思 4. 品德课堂教学与公民教育实践活动的联结点是儿童的现实生活，这包含着( )。 答案：(A)真实的生活(B)普通的生活(C)日常的生活(D)今天的生活（正在进行的生活）(E)首属群体中的生活（儿童主要生活圈中

2013年测绘工作总结及2014年工作计划

2013年测绘工作总结及2014年工作计划 一、2013年测绘工作总结 2013年，公司在各级领导的带领下及各部门员工的支持下，坚持贯彻“技术求新，测绘求精，服务周到，与时俱进”的质量方针，坚持“公正、诚信、专业、快捷”的服务理念，始终把提高业务素质、服务质量和经济效益作为事业发展的根本。在公司全体职工的共同努力下，全年各项工作基本顺利完成。现将我这一年工作做如下总结。 （一）工作作风及理论技术方面 在过去的一年中我身为项目经理，始终都以身作则，带领组员较好的完成领导下达的每项任务，在工作中出现问题能够和领导及同事一起探讨，争取做到每个测绘项目都是合格的。我把我会的毫无保留的与单位同事分享，不会的虚心向领导请教。在这一年得工作中，让我深刻体会到了测绘工作是一项严谨、认真的工作，不负责任，工作敷衍，弄虚作假，是测绘工作坚决不允许的。即使你不会，不懂，也不能装着什么都会了，这样的态度会给公司在社会上造成极坏的影响，我们应该杜绝此类的现象发生，我一直都坚持倡导良好的学习风气，认真的工作态度，领导交代给的任务要按时完成，并且还要保证质量。 测量是一项即艰苦而又技术含量非常高的工作，在这个电子信息的时代，测量设备在不断的更新，大家所掌握的知识也应该要不断的更新。公司的荣辱直接关系到每个员工的切身利益，我们应该看远一点，为单位的长久发展贡献自己微薄之力，只有这样我们才能为公司创造更多的财富与价值，体现自己的专业技术水平，所以在这过去的一年中我报考了全国注册测绘师考试，以使自己能很好

的适应测绘事业的发展，同时我也是想通过自己的努力为公司的发展尽自己的一点微博之力，因为注册测绘师制度将会是以后测绘事业发展的必然，但遗憾的是由于自己资历太浅、经验不足，还有就是准备的不够充分，所以考的不是很理想，但我还会再接再厉，毕竟注册测绘师考试涉及到测量的十二个专业，而我学的仅仅只是其中一个专业的某几个分支，所以在这次的考试中我也颇有收获，至少感觉自己测绘专业理论水平里高了。 （二）业务方面 据统计，在过去的一年中，我项目组较好的完成了20多个开发商房产测绘项目（包括保山交通运输集团有限责任公司腾冲分公司-棚户期改造项目实测；腾冲休闲度假庄园有限公司D、E3、F、G、H、J区项目实测；保丰房地产开发有限责任公司腾冲分公司-玉泉名居-项目实测；南门外文化旅游发展有限公司-月楼、城门楼项目预测；龙龙房地产开发有限公司-凯龙城一期、二期项目实测；云峰置业开发有限公司-云峰山居项目实测；腾恒基房地产开发有限公司-汇和园项目实测；腾龙开发有限公司-龙腾国际项目预测；冲州房地产开发有限公司-天成商业街项目预测；腾冲雅居乐房地产开发有限公司-山居高黎、绿野牧歌、浣溪果林等项目预测；腾冲兴华房地产开发有限公司-兴华物流国际商贸城项目预测；云南腾驿房地产开发有限公司-腾冲国际商贸城项目预测；腾冲恒益东山休闲度假房地产开发有限公司-商业中心、低层住宅组团1、2、4、多层住宅组团1、2、3、6、7项目实测、多层住宅组团4项目预测）；腾冲鼎晟房地产开发有限责任公司-金色家园项目实测；驼峰机场开发管理有限公司-民航大厦项目

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