Diaporthaceae associated with root and crown rot of maize

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© 2011 International Mycological Association

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INTRODUCTION

6RLOERUQH GLVHDVHV VLJQL¿FDQWO\ UHGXFH PDL]H \LHOGV LQ LUULJDWHG V\VWHPV ZKHUH PDL]H IROORZV ZLQWHU ZKHDW LQ WKH .ZD=XOX1DWDO 3URYLQFH RI 6RXWK $IULFD /DPSUHFKW et al. 2YHUWKH\HDUVVHYHUDOIXQJLKDYHEHHQFRQVLVWHQWO\ LVRODWHGIURPPDL]HSODQWVZLWKV\PSWRPVRIFURZQDQGURRW URWLQ¿HOGVVDPSOHGLQ.ZD=XOX1DWDO7KHSDWKRJHQLFLW\RI WKHVHIXQJLUHPDLQVXQUHVROYHG

$PRQJ WKH LVRODWHV REWDLQHG VLQFH  LQ WKH SUHVHQW survey were several coelomycetous fungi with dematiaceous conidia, representing the genera Stenocarpella and

Phaeocytostroma 6XWWRQ  6SHFLHV RI Stenocarpella

DUHFRPPRQO\LVRODWHGIURPGLVHDVHGPDL]HFURSVZRUOGZLGH HVSHFLDOO\GXULQJKXPLGVHDVRQV2GULR]RODet al.7KH two species reported from literature to be associated with FREDQGVWDONURWDQGOHDIEOLJKWRIPDL]HDUHS. macrospora and S. maydis0DUDVDVet al./DWWHUHOO 5RVVL Crous et al.$FFRUGLQJWR6XWWRQDQG:DWHUVWRQ

Diplodia maydisS. maydisFDQDOVRLQIHFWURRWVDQGFDXVH

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et al.  6SHFLHV RI Phaeocytostroma are commonly

DVVRFLDWHG ZLWK VWDON URWV RI GLIIHUHQW KRVWV 6XWWRQ  +ROOLGD\ZLWKP. ambiguum being reported from PDL]H LQ$XVWUDOLD )UDQFH 1RUWK$PHULFD 6HUELD 6WRYROG

et al.0DXULWLXV7DQ]DQLD6XWWRQ6RXWK$IULFD

&URXVet al.DQG<XJRVODYLD/HYLü 3HWURYLü Although S. macrospora and S. maydis have in the past been extensively published as species of Diplodia, 6XWWRQ  SODFHG WKHP LQ Stenocarpella based on their distinct conidiogenesis, a fact supported by later molecular phylogenetic studies, which revealed these taxa to belong to the Diaporthales rather than the Botryosphaeriales &URXVet

al.7KHLUSRVLWLRQ ZLWKLQWKHRUGHUKRZHYHUUHPDLQV

XQUHVROYHG6LPLODUO\P. ambiguum was initially described as a species of SphaeropsisVXJJHVWLQJBotryosphaeriaceae, though nothing is known about the phylogenetic position of

Diaporthaceae associated with root and crown rot of maize

6DQGUD&/DPSUHFKW1_{3HGUR:&URXV}_{-RKDQQHV=*URHQHZDOG}_{<DUHG77HZROGHPHGKLQ}1_{DQG:DOWHU)20DUDVDV} 1_{$JULFXOWXUDO5HVHDUFK&RXQFLO±335,3%DJ;6WHOOHQERVFK6RXWK$IULFDFRUUHVSRQGLQJDXWKRUHPDLOODPSUHFKWV#DUFDJULF]D} _{&%6.1$:)XQJDO%LRGLYHUVLW\&HQWUH8SSVDODODDQ&78WUHFKW7KH1HWKHUODQGV}

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Abstract: Several isolates of coelomycetous fungi with pigmented conidia were consistently isolated from diseased

roots of Zea maysLQLUULJDWHGSORWVPRQLWRUHGLQWKH.ZD=XOX1DWDO3URYLQFHRI6RXWK$IULFD%DVHGRQWKHLUPRUSKRORJ\ WKHVHLVRODWHVFRXOGEHLGHQWL¿HGDVUHSUHVHQWDWLYHRIStenocarpella macrospora, S. maydis, and Phaeocytostroma ambiguum$OWKRXJKVSHFLHVRIStenocarpella are well-known as causal agents of cob and stalk rot and leaf blight of PDL]HLQ6RXWK$IULFDWKHRFFXUUHQFHDQGLPSRUWDQFHRIP. ambiguumLVOHVVZHOOGRFXPHQWHGDQGXQGHUVWRRG7R determine the role of P. ambiguumDVDURRWSDWKRJHQRIPDL]HSDWKRJHQLFLW\WHVWVZHUHFRQGXFWHGXQGHUJODVVKRXVH FRQGLWLRQVDWƒ&QLJKWDQGƒ&GD\WHPSHUDWXUHVXVLQJDSDVWHXULVHGVRLOULYHUVDQGDQGSHUOLWHPHGLXPDQG DVDQGEUDQLQRFXOXP%DVHGRQWKHVHUHVXOWVP. ambiguumZDVVKRZQWREHDSULPDU\SDWKRJHQRIPDL]H but to be less virulent than the positive control, S. maydis)XUWKHUPRUHWRFODULI\WKHKLJKHUOHYHOSK\ORJHQ\RIWKHVH

IXQJDOJHQHUDLVRODWHVZHUHVXEMHFWHGWR'1$VHTXHQFLQJRIWKHQXFOHDUULERVRPDO'1$,76 /683DUWLDOJHQHVHTXHQFHVRIWKHWUDQVODWLRQ HORQJDWLRQIDFWRUDOSKDJHQHZHUHDGGHGWRFRQ¿UPWKHVSHFLHVPRQRSK\O\7RUHVROYHWKHJHQHULFSODFHPHQWRIPhaeocytostroma, additional species such as P. sacchari, P. plurivorum and P. megalosporum were also added to the analysis. Based on these results, Stenocarpella and Phaeocytostroma ZHUH VKRZQ WR EH WZR ZHOO GH¿QHG JHQHUD EHORQJLQJ WR Diaporthales, Diaporthaceae, being closely allied to Phomopsis Diaporthe$OOWKUHHJHQHUDZHUHDOVRREVHUYHGWRIRUPDOSKDDVZHOODVEHWDFRQLGLDDQGDOWKRXJKWKLVSKHQRPHQRQLVZHOOGRFXPHQWHGIRU Phomopsis and Phaeocytostroma, it is a new observation for Stenocarpella,QVSLWHRIWKHGLIIHUHQFHVLQFRQLGLDOSLJPHQWDWLRQQRVXSSRUWFRXOG be obtained for polyphyly in Diaporthaceae, suggesting that as observed in BotryosphaeriaceaeBotryosphaerialesFRQLGLDOSLJPHQWDWLRQLVQRW informative at the family level in Diaporthales

Article info:6XEPLWWHG-DQXDU\$FFHSWHG)HEUXDU\3XEOLVKHG0DUFK Key words: Diplodia diplodiosis Phaeocytostroma phylogeny Stenocarpella systematics Zea mays

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Table 1. &ROOHFWLRQGHWDLOVDQG''%-(0%/*HQ%DQNDFFHVVLRQQXPEHUVRI Phaeocytostroma and Stenocarpella LVRODWHVIRUZKLFKQRYHOVHTXHQFHVZHUHJHQHUDWHGLQWKLVVWXG \ Species Strain no. 1 Substrate Country Collector EMBL

accession no. (ITS, LSU, TEF)

2 Phaeocytostroma ambiguum &3&=) Zea mays South Africa 6/DPSUHFKW )5²)5 &3&=& Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&= 9 Zea mays South Africa 6/DPSUHFKW )5²)5 &3&=5 Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&== Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&== Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&=$% Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&== Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&=+ Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&=) Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&=: Zea mays South Africa 6/DPSUHFKW )5²)5 P. megalosporum &%6,0, Rice-field soil India ² )5)5)5 P. plurivorum &%6 836& Helianthus annuus 3RUWXJDO ² )5)5)5 P. sacchari &%6 ² Japan ² )5)5)5 Stenocarpella macrospora &%6 05& 5DLQGDPDJHG%WPDL]HK\EULGVHDVR Q South Africa -5KHHGHU )5'4² &3&  Zea mays South Africa 3&DOGZHOO )5²² S. maydis &%6 05& %WPDL]HK\EULGIURPVHDVRQ South Africa -5KHHGHU )5'4² &%6 05& 7 UDGLWLRQDOODQGUDFHPDL]HIURPVHD VRQ South Africa -5KHHGHU )5'4)5 &%6 05& &RPPHUFLDOPDL]HK\EULG3 $1IURP VHDVRQ South Africa -5KHHGHU )5'4)5 &3&=) Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&=$% Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&=5 Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&=. Zea mays South Africa 6/DPSUHFKW )5)5)5 &3&=$' Zea mays South Africa 6/DPSUHFKW )5)5 )5 &3&=$( Zea mays South Africa 6/DPSUHFKW )5)5 )5 &3&=3 Zea mays South Africa 6/DPSUHFKW )5)5 )5 &3&=. Zea mays South Africa 6/DPSUHFKW )5)5 )5 &3&=$$ Zea mays South Africa 6/DPSUHFKW )5)5 )5 &3&=% Zea mays South Africa 6/DPSUHFKW )5)5 )5 &3&=' Zea mays South Africa 6/DPSUHFKW )5)5 )5 &3&=& Zea mays South Africa 6/DPSUHFKW )5)5 )5 &3&=) Zea mays South Africa 6/DPSUHFKW )5)5 )5 1&%6 &%6.1$ : )XQJDO %LRGLYHUVLW\ &HQWUH 8WUHFKW 7KH 1HWKHUOD QGV &3& &XOWXUH FROOHFWLRQ RI 3:  &URXV KRXVHG DW &%6 ,0, ,QWHUQDWLRQDO 0\FRORJLFDO ,QVWLWXWH &$%,%LRVFLHQFH (JKDP %DNHKDP /DQH 8. 05& 0HGLFDO 5HVHDUFK &RXQFLO )XVDULXP &ROO HFWLRQ 7\JHUEHUJ 6RXWK$IULFD 836& 8SSVDOD 8QLYHUVLW\ &XOWX UH &ROOHFWLRQ RI )XQJL %RWDQLFDO 0XVHXP 8QLYHUVLW\ RI 8SSVDOD  8SSVDOD6ZHGHQ ,76,QWHUQDOWUDQVFULEHGVSDFHUVDQGWRJHWKHUZLWK6QU '1$/686QU'1$ 7()SDUWLDOWUDQVODWLRQHORQJDWLRQIDFWRU DOSKD

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the genus Phaeocytostroma, and it is generally regarded as

Ascomycota incertae sedis 0\FR%DQNRUJ!)XUWKHUPRUH DOWKRXJKSDWKRJHQLFLW\KDVEHHQFRQ¿UPHGIRU6RXWK$IULFDQ isolates of S. maydis and S. macrosporaRQPDL]H0DUDVDV 9DQGHU:HVWKXL]HQ.HOOHUPDQet al.5KHHGHU

et al.  WKLV KDV QRW EHHQ GRQH IRU P. ambiguum7KH

aim of the present study was thus to resolve the higher order phylogeny of Stenocarpella and Phaeocytostroma, and also determine the importance of P. ambiguum as pathogen on PDL]HZKHQFRPSDUHGWRS. maydis, which is regarded as DQLPSRUWDQWSDWKRJHQRIWKLVKRVW

MATERIALS AND METHODS

Isolates

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DNA phylogeny

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Taxonomy

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Pathogenicity trial

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The pathogenicity trial was conducted in a glasshouse ƒ&QLJKWDQGƒ&GD\WHPSHUDWXUHVXVLQJSODVWLFSRWV  FP GLDP ZLWK D KROGLQJ FDSDFLW\ RI   J SODQWLQJ PHGLXP7KHSODQWLQJPHGLXPZDVPDGHXSRIHTXDODPRXQWV RI VRLO SHUOLWH DQG VDQG ZKLFK ZDV SDVWHXULVHG  PLQ DW  ƒ& DQG OHIW IRU  G EHIRUH EHLQJ PL[HG ZLWK LQRFXOXP $Q LQRFXOXP FRQFHQWUDWLRQ RI   ZWZW ZDV XVHG7KH inoculum was mixed with the planting medium and pots were watered and left to stand overnight in the glasshouse before EHLQJSODQWHGWRPDL]HVHHGVFY3+,'%WKHQH[W GD\ 0DL]H VHHGV ZHUH WUHDWHG ZLWK KRW ZDWHU DW  ƒ& IRU  PLQ 'DQLHOV  WR HQVXUH WKDW FOHDQ VHHG ZDV XVHG 3RWV ZHUH ZDWHUHG HYHU\ DOWHUQDWH GD\ WR ¿HOG FDSDFLW\ 3DWKRJHQLFLW\ DQG UHODWLYH YLUXOHQFH RI HDFK LVRODWH ZHUH determined by calculating the percentage survival and plant JURZWKVKRRWOHQJWKDVZHOODVWKHSHUFHQWDJHSODQWVZLWK FURZQDQGURRWURWVHYHULW\XVLQJD±VFDOHZLWK QRURRW URW !±URRWURW !±URRWURW !± URRWURWDQG !±URRWURWZNDIWHUSODQWLQJ7R FRQ¿UPWKHSUHVHQFHRIWKHGLIIHUHQWIXQJLUHLVRODWLRQVZHUH PDGHE\SODWLQJPPSLHFHVRIWLVVXHH[FLVHGIURPFURZQV and roots of plants with crown and root rot representatively VHOHFWHG IURP HDFK WUHDWPHQW RQ 3'$ 7KH H[SHULPHQWDO design was a randomised block design with three replicates IRUHDFKWUHDWPHQW

Statistical analysis

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Magnaporthe grisea AB026819

Gaeumannomyces graminis var. avenae AF362556 Coniochaetidium savoryi AY346276

Coniochaeta sp. AY346275 Coniochaeta velutina EU999180

Calosphaeria pulchella AY761075

Phaeoacremonium sphinctrophorum DQ173151 Togninia novae-zealandiae AY761081 Asterosporium asterospermum AB553741 Asterosporium asterospermum AB553745

Mazzantia napelli AF408368

Valsa ceratosperma AF408386 Leucostoma niveum AF408367

Valsella adhaerens AF408388 Greeneria uvicola AF362570

Melanconiella spodiaea AF408370 Cryphonectria macrospora AF408340

Endothiella gyrosa AF362555 Cryphonectria nitschkei AF408341

Coniella australiensis AF408336 Pilidiella granati AF408379

Schizoparme botrytidis AF408383 Harknessia eucalypti AF408363

Wuestneia molokaiensis AF408390 Harknessia gibbosa EF110615

Ophiovalsa betulae AF408375 Gnomonia setacea AF362563 Phragmoporthe conformis AF408377 Melanconis stilbostoma AF408374

Melanconis alni AF362566 Melanconis marginalis AF408373 Diaporthe padi AF408354

Diaporthe pustulata AF408358 Diaporthe perjuncta AF408356

Diaporthe decedens AF408348 Diaporthe detrusa AF408349 Phomopsis sclerotioides AF439631 Phomopsis asparagi AF439634

Diaporthe medusaea AF362560 Diaporthe pardalota AF408355 Diaporthe eres AF408350 Diaporthe oncostoma AF408353 Phomopsis vaccinii AF439630 Diaporthe angelicae AY196781 Stenocarpella macrospora DQ377934 Phaeocytostroma plurivorum CBS 113835 Phaeocytostroma megalosporum CBS 284.65 Phaeocytostroma sacchari CBS 275.34 Phaeocytostroma ambiguum CPC 16776 Phaeocytostroma ambiguum CPC 17074 Phaeocytostroma ambiguum CPC 17075 Phaeocytostroma ambiguum CPC 17076 Phaeocytostroma ambiguum CPC 17077 Phaeocytostroma ambiguum CPC 17078 Phaeocytostroma ambiguum CPC 17079 Phaeocytostroma ambiguum CPC 17072 Stenocarpella maydis DQ377935 Stenocarpella maydis CPC 16778 Stenocarpella maydis CPC 16781 Stenocarpella maydis CPC 16777 Stenocarpella maydis CPC 16779 Stenocarpella maydis CPC 16780 Stenocarpella maydis CPC 16782 Stenocarpella maydis CPC 16784 Stenocarpella maydis CPC 16785 Stenocarpella maydis CPC 16786 Stenocarpella maydis CPC 16787 Stenocarpella maydis CPC 16788 Stenocarpella maydis CPC 16789 Stenocarpella maydis CPC 17073 Stenocarpella maydis DQ377937 Stenocarpella maydis DQ377936 10 changes Key to Families: 1. Togniniaceae 2. Valsaceae 3. Melanconidiaceae 4. Cryphonectriaceae 5. Gnomoniaceae 6. Diaporthaceae

1

3a

3b

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4

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Family

Coniochaetales Diaporthales 100 100 100 100 100 100 100 100 77 84 98 99 56 61 50 50 59 57 95 95 80 75 93 83 92 84 59 93 95 Fig. 1.7KH¿UVWRIHTXDOO\PRVWSDUVLPRQLRXVWUHHVREWDLQHGIURPDKHXULVWLFVHDUFKZLWKUDQGRPWD[RQDGGLWLRQV3$83YE %RRWVWUDSVXSSRUWYDOXHVDUHVKRZQDWWKHQRGHVDQGVWULFWFRQVHQVXVEUDQFKHVDUHWKLFNHQHG)DPLOLHVDUHLQGLFDWHGLQGLIIHUHQWFRORXUHG ER[HV7KHWUHHZDVURRWHGWRGaeumannomyces graminis YDUavenae *HQ%DQN $)DQGMagnaporthe grisea *HQ%DQN $%

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RESULTS

Phylogenetic analyses

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10 changes

Phomopsis viticola FJ790863

Phaeocytostroma sacchari CBS 275.34 Diaporthe cynaroidis EU552122

Phaeocytostroma megalosporum CBS 284.65 Phaeocytostroma plurivorum CBS 113835 CPC 17072 CPC 17078 CPC 17076 CPC 17079 CPC 17075 CPC 17083 CPC 17077 CPC 16776 CPC 16775 CPC 17074 CPC 17071 GQ259128 CBS 117560 CPC 11863 GQ167224 GQ167225 AY332487 AY332480 GQ167213 GQ167214 GQ167215 GQ167216 CPC 16788 CPC 16787 CPC 16786 CPC 16784 CPC 16782 CPC 16781 CPC 16779 CPC 16778 CPC 16777 CBS 117557 GQ167218 CPC 16789 GQ167219 GQ167220 AY332482 GQ167221 GQ167222 CBS 117558 CBS 117559 GQ167223 GQ259129 GQ259130 CPC 16785 CPC 16780 CPC 17073 Phaeocytostroma ambiguum Stenocarpella macrospora Stenocarpella maydis 100 75 74 70 100 100 100 Fig. 2.7KH¿UVWRIWZRHTXDOO\PRVWSDUVLPRQLRXVWUHHVREWDLQHGIURPDKHXULVWLFVHDUFKZLWKUDQGRPWD[RQDGGLWLRQV3$83YE %RRWVWUDSVXSSRUWYDOXHV!DUHVKRZQDWWKHQRGHVDQGVWULFWFRQVHQVXVEUDQFKHVDUHWKLFNHQHG7KHWKUHHVSHFLHVIURPPDL]HDUH LQGLFDWHGLQGLIIHUHQWFRORXUHGER[HV7KHWUHHZDVURRWHGWRPhomopsis viticola *HQ%DQN )-

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RI WKH /68 UHJLRQ )LJ  VKRZV WKDW Stenocarpella and

Phaeocytostroma are embedded with the Diaporthaceae and

could not be distinguished phylogenetically from Diaporthe 7KHPDQXDOO\DGMXVWHG,76DOLJQPHQWFRQWDLQHGWD[D LQFOXGLQJWKHRXWJURXSVHTXHQFHDQGRIWKHFKDUDFWHUV XVHG LQ WKH SK\ORJHQHWLF DQDO\VLV  ZHUH SDUVLPRQ\ LQIRUPDWLYH  ZHUH YDULDEOH DQG SDUVLPRQ\XQLQIRUPDWLYH DQGZHUHFRQVWDQW7ZRHTXDOO\PRVWSDUVLPRQLRXVWUHHV ZHUHUHWDLQHGIURPWKHKHXULVWLFVHDUFKWKH¿UVWRIZKLFKLV

VKRZQ LQ )LJ  7/   &,   5,   5&  7KHSK\ORJHQHWLFWUHHRIWKH,76UHJLRQ)LJVKRZV WKDW WKH VHTXHQFHV RI VSHFLHV RI Phaeocytostroma form a PRQRSK\OHWLF OLQHDJH ZLWK D ERRWVWUDS VXSSRUW YDOXH RI  ZKHUHDVWKHPRQRSK\OHWLFOLQHDJHIRUStenocarpella was SRRUO\VXSSRUWHGQRWVKRZQRQWUHH

7KHPDQXDOO\DGMXVWHG7()DOLJQPHQWFRQWDLQHGWD[D LQFOXGLQJWKHRXWJURXSVHTXHQFHDQGRIWKHFKDUDFWHUV GXH WR WKH LQFOXVLRQ RI D PXFK VKRUWHU RXWJURXS VHTXHQFH

10 changes

Phomopsis viticola GU294706

Phaeocytostroma plurivorum CBS 113835 CPC 16775 CPC 17074 CPC 17072 CPC 17083 CPC 17079 CPC 17078 CPC 17077 CPC 17071 CPC 17076 CPC 16776 CPC 17075 Phaeocytostroma sacchari CBS 275.34 Phaeocytostroma megalosporum CBS 284.65 CBS 117558 CPC 17073 CPC 16789 CPC 16788 CPC 16787 CPC 16786 CPC 16785 CPC 16784 CPC 16779 CPC 16778 CPC 16777 CBS 117559 CPC 16780 CPC 16782 CPC 16781 Phaeocytostroma ambiguum 100 76 89 100 65 100 Stenocarpella maydis Fig. 3.7KH¿UVWRIWZRHTXDOO\PRVWSDUVLPRQLRXVWUHHVREWDLQHGIURPDKHXULVWLFVHDUFKZLWKUDQGRPWD[RQDGGLWLRQV3$83YE %RRWVWUDSVXSSRUWYDOXHV!DUHVKRZQDWWKHQRGHVDQGVWULFWFRQVHQVXVEUDQFKHVDUHWKLFNHQHG7KHWZRVSHFLHVIURPPDL]HIRUZKLFK 7()VHTXHQFHVZHUHDYDLODEOHDUHLQGLFDWHGLQGLIIHUHQWFRORXUHGER[HV7KHWUHHZDVURRWHGWRPhomopsis viticola *HQ%DQN *8

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FRPSDUHG WR WKH OHQJWK RI WKH LQJURXS VHTXHQFHV XVHG LQ WKH SK\ORJHQHWLF DQDO\VLV  ZHUH SDUVLPRQ\LQIRUPDWLYH  ZHUH YDULDEOH DQG SDUVLPRQ\XQLQIRUPDWLYH DQG  ZHUH FRQVWDQW 7ZR HTXDOO\ PRVW SDUVLPRQLRXV WUHHV ZHUH UHWDLQHGIURPWKHKHXULVWLFVHDUFKWKH¿UVWRIZKLFKLVVKRZQ LQ)LJ7/ &, 5, 5& 7KH SK\ORJHQHWLFWUHHRIWKH7()UHJLRQ)LJVKRZVYHU\OLWWOH LQWUDVSHFL¿FYDULDWLRQIRUS. maydis and P. ambiguum

Taxonomy

Phaeocytostroma ambiguum 0RQW 3HWU Feddes

Repert42

Basionym: Sphaeropsis ambigua0RQWAnn. Sci. Nat.,

Bot12

Synonyms: Phaeocytostroma istrica 3HWU Ann. Mycol

19

Phaeocytosporella zeae Stout, Mycologia 22 



()LJ

Fig. 4. Phaeocytostroma ambiguum &3&  A. &RQLGLRPDWD RQ SRWDWRGH[WURVH DJDU B, C. &RQLGLRPDWD RQ SLQH QHHGOH DJDU D–F.

&RQLGLRSKRUHVDQGSDUDSK\VHVG.+\DOLQHFRQLGLRSKRUHVJLYLQJULVHWREURZQFRQLGLDOPDVVH, I.$OSKDFRQLGLDJ. Conidiogenous cells giving ULVHWREHWDFRQLGLDK.%HWDFRQLGLD6FDOHEDUV —P

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ConidiomataRQ31$DQG2$LPPHUVHGLQLWLDOO\VROLWDU\EXW IRUPLQJDVWURPDXSWRPPGLDPEHFRPLQJPXOWLORFXODUZLWK RQHWRVHYHUDOFOHDUO\GH¿QHGEODFNQHFNVH[WHQGLQJDERYH WKHVWURPDXSWR—PWDOODIWHUZNEXWEHFRPLQJPRUH HORQJDWHGZLWKDJHXSWR—PZLGHZLWKWHUPLQDORVWLROH XS WR  —P ZLGH Conidiomatal wall black, consisting of several layers of textura intricata to textura angularis, up to  —P ZLGH IRUPLQJ DQ LQQHU SDOH EURZQ WR K\DOLQH OD\HU XS WR  —P ZLGH Alpha conidiophores tightly aggregated, subcylindrical, branched in mid region, consisting of ± VXSSRUWLQJ FHOOV JLYLQJ ULVH WR VHSWDWH F\OLQGULFDO FRQLGLRJHQRXV FHOOV RU SDUDSK\VHV ±VHSWDWH ± î ± —P Alpha conidiogenous cells hyaline, subcylindrical, WHUPLQDO DQG ODWHUDO ± î ± —P DSH[ ZLWK PLQXWH SHULFOLQDOWKLFNHQLQJDQGFROODUHWWHParaphyses intermingled between conidiophores or arising from same conidiophores that give rise to conidiogenous cells, subcylindrical, hyaline, EUDQFKHG RU QRW ± WUDQVYHUVHO\ VHSWDWH ± î ± —P DSH[ EOXQWO\ URXQGHG Alpha conidia medium brown, VPRRWK HOOLSVRLG WR S\ULIRUP VRPHZKDW FODYDWH RQ 31$ widest in middle of conidium, apex bluntly rounded, base WUXQFDWH ±±± î ±±± —P Beta

conidiophores interspersed among alpha conidiophores,

K\DOLQHVXEF\OLQGULFDOEUDQFKHG±VHSWDWH±î± —P Beta conidiogenous cells phialidic, integrated, terminal DQG ODWHUDO ± î ± —P Beta conidia subcylindrical, straight to slightly curved, hyaline, smooth, widest in middle, WDSHULQJ WR DFXWHO\ URXQGHG DSH[ EDVH WUXQFDWH ± î ±—P

Culture characteristics: Colonies RQ2$ÀDWVSUHDGLQJZLWK

VPRRWK PDUJLQV DQG VSDUVH DHULDO P\FHOLXP VXUIDFH ÀDW ZLWK D GXOO EODFN OD\HU DQG SDWFKHV RI ÀDW ZKLWH P\FHOLXP IRUPLQJDOD\HURQWKHVXUIDFHFRYHULQJWKHSODWHZLWKLQZN 2Q3'$VLPLODUH[FHSWWKDWWKHEODFNOD\HUH[WHQGVIURPWKH centre outwards, with the white layer in the outer region, less GHQVHWKDQRQ2$UHYHUVHGXOOEODFNLQPLGGOHSDOHZKLWH LQRXWHUUHJLRQ2Q0($DSSHDULQJROLYDFHRXVEODFNGXHWR ZRROO\JUH\DHULDOP\FHOLXPUHYHUVHVLPLODUDVRQ3'$

Specimens examined: FRANCE ?: from stems of Zea mays 3&

 ± KRORW\SH SOUTH AFRICA .ZD=XOX1DWDO :LQWHUWRQ

*RXUWRQ IDUP RQ URRWV RI Zea mays  S. Lamprecht &%6 + ± epitypus hic designatus FXOWXUH H[HSLW\SH &3&  &%6

Notes: The beta conidia described above for P. ambiguum

ZHUHUHFHQWO\UHSRUWHGE\/HYLü 3HWURYLüDQGVHHP WREHFRPPRQO\SURGXFHGE\LVRODWHVRIWKLVVSHFLHV2WKHU taxa in the Diaporthaceae )LJV  , such as Phomopsis Diaporthe DOVR SURGXFH EHWD FRQLGLD VXJJHVWLQJ WKDW the putative link between Phaeocytostroma iliau and

Clypeoporthe iliau%DUUFRXOGEHFRUUHFW

Stenocarpella maydis

%HUN

%

6XWWRQ

Coelomycetes

Basionym: Sphaeria maydis %HUN Hooker’s J. Bot.,

London 6

Synonyms: Additional synonyms are listed in Sutton



)LJ

Specimens examined: SOUTH AFRICA.ZD=XOX1DWDO6LPGODQJHQWVKD

Bt Zea mays K\EULG IURP  VHDVRQ J. Rheeder H[HSLW\SH &%6   05&  GHVLJQDWHG LQ &URXV et al.  LELG &%6   05&  +ODELVD FRPPHUFLDO K\EULG 3$1 05& &%6

Note: Conidia subcylindrical to narrowly ellipsoid, straight,

FXUYHG RFFDVLRQDOO\ LUUHJXODU ±VHSWDWH VPRRWKZDOOHG SDOH EURZQ DSH[ REWXVH EDVH WUXQFDWH ± î ± —P 6XWWRQ

Stenocarpella macrospora (DUOH%6XWWRQMycol.

Pap141

Basionym: Diplodia macrospora Earle, Bull. Torrey Bot.

Cl24

Synonyms: Additional synonyms are listed in Sutton



)LJ

Fig. 5. Stenocarpella maydis &%6A.&RQLGLRPDZLWKH[XGLQJEODFNFRQLGLDOFLUUKXVRQSLQHQHHGOHDJDUB. Conidiogenous cells giving

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Specimens examined: SOUTH AFRICA .ZD=XOX1DWDO +ODELVD UDLQ

damaged Bt Zea mays K\EULG  VHDVRQ J. Rheeder H[ HSLW\SH&%6 05&GHVLJQDWHGLQ&URXVet al. .ZD=XOX1DWDOZea maysNHUQHOVP. Caldwell&3&  &%6

Notes: Conidia subcylindrical to narrowly ellipsoid, straight,

FXUYHG RFFDVLRQDOO\ LUUHJXODU ±VHSWDWH VPRRWKZDOOHG SDOH EURZQ DSH[ REWXVH EDVH WUXQFDWH ± î ± —P 6XWWRQ  6HYHUDO FXOWXUHV DOVR IRUPHG K\DOLQH scolecosporous, curved beta conidia, which is a new observation for S. macrospora, but not uncommon in the

Diaporthaceae)LJ

Pathogenicity trial

Stenocarpella maydis VLJQL¿FDQWO\ UHGXFHG WKH VXUYLYDO RI

seedlings compared to the control and P. ambiguum7DEOH  Stenocarpella maydis LVRODWHV =) =$% =5 =' DQG =& VLJQL¿FDQWO\ UHGXFHG VHHGOLQJ VXUYLYDO compared to the control, with the lowest survival rates UHFRUGHGIRU=%=5DQG='7DEOH

Both P. ambiguum and S. maydis FDXVHG VLJQL¿FDQWO\ more crown and root rot, and growth reduction, than the FRQWURO+RZHYHUS. maydis was the most virulent, causing VLJQL¿FDQWO\ PRUH FURZQ DQG URRW URW DQG JURZWK UHGXFWLRQ than P. ambiguum 7DEOH  2I WKH LVRODWHV LQFOXGHG LQ this study, P. ambiguum LVRODWHV =9 == =$% =&DQG=:DQGDOOS. maydisLVRODWHVH[FHSW=% VLJQL¿FDQWO\UHGXFHGSODQWJURZWKVKRRWOHQJWK7KHKLJKHVW growth reductions were recorded for S. maydisLVRODWHV=5 DQG =' EXW JURZWK UHGXFWLRQ FDXVHG E\ WKHVH LVRODWHV GLGQRWGLIIHUVLJQL¿FDQWO\IURPWKDWFDXVHGE\LVRODWHV=3 DQG=&$OOLVRODWHVRIERWKIXQJLFDXVHGVLJQL¿FDQWFURZQ rot compared to the control, except for P. ambiguum isolates =5===+DQG=$6DQGDOOLVRODWHVWHVWHG H[FHSW=+P. ambiguumFDXVHGVLJQL¿FDQWURRWURW7KH KLJKHVWURRWURWVHYHULWLHVZHUHUHFRUGHGIRULVRODWHV=5 =3='DQG=&7DEOH

Fig. 6. Stenocarpella macrospora &3&A.&RQLGLRPDZLWKH[XGLQJFRQLGLDOPDVVRQSLQHQHHGOHDJDUB, C. Conidiogenous cells giving

ULVHWRFRQLGLDD.+\DOLQHOD\HURIFRQLGLRJHQRXVFHOOVJLYLQJULVHWREURZQFRQLGLDOPDVVE, F.$OSKDFRQLGLDG. Conidiogenous cells giving rise WREHWDFRQLGLDH.%HWDFRQLGLD6FDOHEDUV —P

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DISCUSSION

Stenocarpella maydisLVZHOOGRFXPHQWHGDVDPDMRUFDXVH

RIFREURWRIPDL]H8OOVWUXSDQGWKHFKLHIRUJDQLVP DVVRFLDWHG ZLWK GLSORGLRVLV .HOOHUPDQ et al.  ,Q contrast, S. macrospora has been seen as of less importance when compared to S. maydis9LUWDQHQet al.,Q/DWLQ America and Africa, however, both pathogens have been

regarded as important ear-rotting pathogens, because of their ability to produce toxins in infected grain, which may EHXVHGWRIHHGOLYHVWRFNDQGSRXOWU\0DUDVDVet al. $ODWHUVWXG\E\/DWWHUHOO 5RVVLKRZHYHUSURGXFHG UHVXOWV FRQWUDGLFWRU\ WR WKRVH RI +RSSH  DFWXDOO\ suggesting that S. macrospora was more virulent on young stalks than isolates of S. maydis 7KH FRQWUDVWLQJ UHVXOWV were partially explained by the fact that there may be strains

Table 2. 6XUYLYDOVKRRWOHQJWKDQGFURZQDQGURRWURWUHFRUGHGIRUPDL]HVHHGOLQJVLQRFXODWHGZLWKPhaeocytostroma ambiguum and Steno-carpella maydisXQGHUJODVVKRXVHFRQGLWLRQV

Fungus Survival (%)x _{Shoot length (mm)} x _{Crown rot (%)}x y _{Root rot severity} x z

Control D D F F

P. ambiguum D E E E

S. maydis E F D D

x_{0HDQVZLWKLQDFROXPQIROORZHGE\WKHVDPHOHWWHUGRQRWGLIIHUVLJQLILFDQWO\3 } y_{3HUFHQWDJHSODQWVZLWKFURZQURW}

]_{5RRWURWVHYHULW\UDWHGRQDVFDOHRI±ZLWK QRURRWURW !±URRWURW !±URRWURW !±DQG!±}

URRWURW

Table 3. Effect of different isolates of Phaeocytostroma ambiguum and Stenocarpella maydisRQVXUYLYDOSODQWJURZWKVKRRWOHQJWKDQG

FURZQDQGURRWURWRIPDL]HVHHGOLQJVXQGHUJODVVKRXVHFRQGLWLRQV

Fungus Isolate Survival (%)x _{Shoot length (mm)} x _{Crown rot (%)}x y _{Root rot severity} x z

Control D DE M K

P. ambiguum =9 D GHIJ GHI J

=) D EFG FG J == D FG KLM J == D DEFG JKL J =$% D FGHI D I == D D KLM J =+ D DE KLM K =$6 DE D KLM J =) D DEFG LM J

=& D IJK D GHI

=: DE FGH D HI

S. maydis =) EF HIJK D HI

=$% H JK GH GF

=5 GH M D E

=. DEF IJK EF GHI

=$' D IJK EF HI

=3 DEF LM FG EF

=. DEF KL D GH

=$' DEF FGHI IJK HI

=% D DEF HIJ J

=' H M JKL DE

=& FG LM DE D

x_{0HDQVZLWKLQDFROXPQIROORZHGE\WKHVDPHOHWWHUGRQRWGLIIHUVLJQLILFDQWO\3 } y_{3HUFHQWDJHSODQWVZLWKFURZQURW}

]_{5RRWURWVHYHULW\UDWHGRQDVFDOHRI±ZLWK QRURRWURW !±URRWURW !±URRWURW !±DQG !±}

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ZLWK GLIIHULQJ YLJRXU ZLWKLQ HDFK VSHFLHV ,Q VSLWH RI WKHLU virulence, S. maydis is more commonly observed in the USA /DWWHUHOO 5RVVLDVZHOODV6RXWK$IULFD0DUDVDVet

al.$OWKRXJKFRPPRQO\DVVRFLDWHGZLWKURRWDQGVWDON

URWRIPDL]HQRWPXFKLVNQRZQDERXWWKHSDWKRJHQLFLW\RI

P. ambiguum, other than the study by Stovold et al.

LQ $XVWUDOLD ,WV SRWHQWLDO UROH DV SULPDU\ SDWKRJHQ ZDV KRZHYHU FRQ¿UPHG LQ WKH SUHVHQW VWXG\ WKRXJK VWUDLQV RI

P. ambiguum generally appeared to be less virulent than the

strains of S. maydis WHVWHG 7DEOHV   1HYHUWKHOHVV P.

ambiguum should be considered as an important pathogen of

PDL]HDQGFHUWDLQO\DVSDUWRIDVRLOERUQHGLVHDVHFRPSOH[ FRXOGUHVXOWLQVLJQL¿FDQWGDPDJHWRPDL]HSODQWV6XUYH\V FRQGXFWHG IRU D QXPEHU RI VHDVRQV LQ WKH .ZD=XOX1DWDO province showed that the incidences of both fungi increase VLJQL¿FDQWO\ WRZDUGV WKH HQG RI WKH JURZLQJ VHDVRQ ZKHQ PDL]HSODQWVDUHRIWHQVXEMHFWHGWRPRLVWXUHVWUHVV5HVXOWV QRW VKRZQ 6WRYROG et al.  UHSRUWHG WKDW ZKLOH P.

ambiguumFDQFDXVHH[WHQVLYHLQIHFWLRQRIPDL]HURRWVWKH

IXQJXVGLGQRWVLJQL¿FDQWO\DIIHFWWKHJURZWKRISODQWVXQGHU RSWLPDO FRQGLWLRQV RI VRLO PRLVWXUH DQG QXWULWLRQ $OWKRXJK WKHVH IXQJL PD\ RYHUZLQWHU LQ LQIHFWHG PDL]H UHVLGXH IURP where they infect the roots, mesocotyl, crown and eventually the stalks of new plants, not much is known about their host VSHFL¿FLW\ DQG ZKHWKHU WKH\ FRXOG DOVR EH LVRODWHG IURP JUDVVHVWKDWJURZLQWKHYLFLQLW\RIPDL]H¿HOGV

Based on their pigmented conidia and Diplodia-like morphology, both Stenocarpella and Phaeocytostroma have in the past been suspected to be members of the

Boytyosphaeriaceae, being initially described in genera

such as Diplodia and Sphaeropsis +RZHYHU &URXV et al. UHYHDOHGStenocarpella to belong to the Diaporthales, though the phylogenetic relationships of Phaeocytostroma UHPDLQHG REVFXUH XQWLO WKH SUHVHQW VWXG\ )URP WKH WD[D WUHDWHGKHUH)LJVLWLVFOHDUWKDWERWKDQDPRUSKJHQHUD are best allocated to the Diaporthales, Diaporthaceae This is somewhat surprising, as their pigmented conidia suggests that they might represent a separate family within the Diaporthales,QVSLWHRIWKHVHGLIIHUHQFHVKRZHYHUQR support could be obtained for polyphyly in Diaporthaceae 7KHVH ¿QGLQJV VXJJHVW WKDW DV REVHUYHG HDUOLHU LQ WKH

BotryosphaeriaceaeBotryosphaeriales&URXVet al.

3KLOOLSV et al.  FRQLGLDO SLJPHQWDWLRQ DSSHDUV WR EH uninformative at the family level, while conidiogenesis, and the ability to produce both alpha and beta conidia, appear more informative at family level in Diaporthaceae Diaporthales

ACKNOWLEDGEMENTS

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0DUDVDV :)2 :HVWKXL]HQ *&$ YDQ GHU  Diplodia macrosporaWKHFDXVHRIOHDIEOLJKWDQGFREURWRIPDL]HZea maysLQ6RXWK$IULFDPhytophylactica 11±

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2GULR]ROD ( 2GHyQ &DQWRQ * &OHPHQWH * (VFDQGH $  Diplodia maydis D FDXVH RI GHDWK RI FDWWOH LQ$UJHQWLQD New Zealand Veterinary Journal 53±

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