Genotypic Diversity and Host-Specificity of Frankia Bacteria Associated with Sympatric Populations of Alnus Rubra and Alnus Rhombifolia in Oregon

Genotypic Diversity and Host-Specificity of Frankia Bacteria Associated with Sympatric Populations of Alnus rubra and Alnus rhombifolia in Oregon

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(IZ[YHJ[ %LRORJLFDOQLWURJHQ¿[DWLRQLVRQHRIWKHPRVWFULWLFDOSURFHVVHVFRQWULEXWLQJWRHFRV\VWHPSURGXFWLYLW\DQGVWDELOLW\RQD JOREDOVFDOH,QWHPSHUDWHFOLPDWHVRIWKHQRUWKHUQKHPLVSKHUHSODQWURRWDVVRFLDWHGEDFWHULDRIWKHJHQXVFrankia are the PDMRUQLWURJHQ¿[HUVLQIRUHVWHQYLURQPHQWV7UHHVEHORQJLQJWRWKHJHQXVAlnus are the most widespread hosts of Frankia LQWKH3DFL¿F1RUWKZHVWDQGDP\ULDGRIELRWLFDQGDELRWLFIDFWRUVFDQLQÀXHQFHWKHUREXVWQHVVRIWKLVV\PELRVLV+RVW LGHQWLW\DQGEDFWHULDOVWUDLQDUHLPSRUWDQWIHDWXUHVWKDWFDQLPSDFWAlnus-FrankiaDVVRFLDWLRQEXWOLWWOHLVNQRZQDERXW WKHLQWHUSOD\RILQWUDJHQHULFKRVWVWKDWFRRFFXULQQDWXUDOVHWWLQJV,QWKLVVWXG\ZHLQYHVWLJDWHGWKHJHQHWLFGLYHUVLW\DQG KRVWVSHFL¿FLW\RIFrankia EDFWHULDDVVRFLDWHGZLWKV\PSDWULFDOO\RFFXUULQJSRSXODWLRQVRIAlnus rubra UHGDOGHU DQG Alnus rhombifolia ZKLWHDOGHU LQ2UHJRQ%DVHGRQVHTXHQFHDQDO\VLVRIWKHnifHJHQHUHFRYHUHGIURPURRWQRGXOHVZH IRXQGORZRYHUDOOEDFWHULDOGLYHUVLW\2QHGRPLQDQWFrankiaJHQRW\SHZDVDVVRFLDWHGZLWKERWKKRVWVSHFLHVLQGLFDWLQJ DODFNRIVWURQJKRVWVSHFL¿FLW\LQWKLVV\VWHP2XUUHVXOWVVXJJHVWWKDWFHUWDLQLQWUDJHQHULFSODQWKRVWVZLWKRYHUODSSLQJ GLVWULEXWLRQVVKRZFURVVFRPSDWLELOLW\ZLWKV\PELRWLFDFWLQRUKL]DOEDFWHULDDQGWKDWORZVWUDLQGLYHUVLW\RIWKHVHEDFWHULD FDQSHUVLVWDFURVVPL[HGKRVWSRSXODWLRQV

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Diversity of Frankia in Alnus

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DNA Extraction '1$ZDVH[WUDFWHGIURPLQGLYLGXDO nodule lobes using the Sigma Tissue ([WUDFW1$PS.LW 6LJPD$OGULFK 6W/RXLV02 )RUW\ȝ/RIH[WUDF WLRQEXIIHUZHUHDGGHGLQWRHDFK mL microcentrifuge tube contain LQJDQLQGLYLGXDOQRGXOHOREHZKLFK ZDVWKHQKRPRJHQL]HGXVLQJDVWHULOH micropestle. Lobe homogenates were FHQWULIXJHGIRUPLQXWHDWUSP [J DQGȝ/RIVXSHUQDWDQW from each tube transferred into an LQGLYLGXDOP/3&5VWULSWXEH 6WULSWXEHVFRQWDLQLQJH[WUDFWLRQV ZHUHSODFHGLQDWKHUPDOF\FOHUDQG LQFXEDWHGDW&IRUPLQXWHV )LJXUH 0DSRIFROOHFWLRQVLWHLQZHVWHUQ2UHJRQGLVSOD\LQJORFDWLRQVRI IROORZHGE\&IRUPLQXWHV LQGLYLGXDOAlnus rubra and A. rhombifolia trees. Alnus rubra trees are ODEHOOHGZLWKVWDULFRQVA. rhombifolia trees with circles. Information )ROORZLQJLQFXEDWLRQȝ/RIQHX on the associated Frankia JHQRW\SHUHFRYHUHGIURPURRWQRGXOHVLV WUDOL]DWLRQEXIIHUZHUHSLSHWWHGLQWR SURYLGHGIRUHDFKWUHH,FRQVZLWKRXWFRUUHVSRQGLQJODEHOVLQGLFDWH HDFKVDPSOHZKLFKZHUHWKHQEULHÀ\ trees from which nifHVHTXHQFHVZHUHQRWREWDLQHG YRUWH[HGDQGVWRUHGDW&XQWLOXVH

PCR and Sequencing Surface Sterilization $ES EDVHSDLU SRUWLRQRIWKHnifH gene was DPSOL¿HGE\3&5XVLQJFrankia-VSHFL¿FSULPHUV 5RRWVDQGDWWDFKHGQRGXOHVZHUHZDVKHGZLWK QLI+I ƍ**&$$*7&&$&&$&&&$*&ƍ GHLRQL]HGZDWHUWRUHPRYHVRLODQGRUJDQLFPDW DQGQLI+U ƍ&7&*$7*$&&*7&$7&&**& WHU1RGXOHVZHUHWKHQUHPRYHGIURPURRWVZLWK ƍ 3&5UHDFWLRQVZHUHVHWXSLQȝ/YROXPHV DVWHULOHVFDOSHODQGSODFHGLQWRLQGLYLGXDOP/ FRQWDLQLQJȝ/3&5ZDWHUȝ/*R7DT VWHULOHVFLQWLOODWLRQYLDOV(DFKYLDOZDVWKHQ¿OOHG 0DVWHU0L[ȝ/%6$ PJP/ ȝ/ ZLWKP/RIEOHDFK K\SRFKORULWH RIHDFKSULPHU ȝ0 DQGȝ/RIGLOXWHG solution and agitated in an orbital shaker at room WHPSODWH5HDFWLRQPL[WXUHVZHUHWKHQVXEMHFWHG WHPSHUDWXUH & IRUPLQXWHVDWUSP WRWKHIROORZLQJWKHUPDOF\FOLQJFRQGLWLRQV& $IWHUDJLWDWLRQWKHEOHDFKVROXWLRQZDVGHFDQWHG IRUPLQXWHVF\FOHVDW&IRUVHFRQGV IURPHDFKYLDODQGWKHEOHDFKZDVKLQJSURFHVVZDV &IRUVHFRQGV&IRUVHFRQGVDQG

Balkan et al.

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Diversity of Frankia in Alnus

'RZQORDGHG)URPKWWSVELRRQHRUJMRXUQDOV1RUWKZHVW6FLHQFHRQ-DQ 7erms of Use: https://bioone.org/terms-of-use $FFHVVSURYLGHGE\3RUWODQG6WDWH8QLYHUVLW\ 7$%/( $EXQGDQFHRIFrankia JHQRW\SHVDVVRFLDWHGZLWKKRVWAlnus species. Discussion Frankia*HQRW\SH +RVW6SHFLHV 7KHJRDORIWKHFXUUHQWVWXG\ZDV '-%) '-%) '-%) '-%) '-%) Alnus rubra WRDVVHVVJHQHWLFGLYHUVLW\DQG Alnus rhombifolia KRVWVSHFL¿FLW\RIDFWLQRUKL]DO Frankia bacteria associated with ,GHQWLFDOWRJHQRW\SH./ *HQ%DQNDFFHVVLRQ*8 GHVFULEHGLQ.HQ WKHURRWVRIWZRFRRFFXUULQJ QHG\HWDO AlnusWD[D2XU¿QGLQJVVXJJHVW ,GHQWLFDOWRJHQRW\SH./ *HQ%DQNDFFHVVLRQ*8 GHVFULEHGLQ .HQQHG\HWDO WKDWKRVWLGHQWLW\LVQRWDVWURQJ GULYHURIFrankiaJHQRW\SHDEXQ GDQFHDVVRFLDWHGZLWKV\PSDWULF ¿YHFrankia JHQRW\SHVVKDUHGE\WKHWZRAlnus populations of Alnus rubra and A. rhombifolia. SRSXODWLRQVLOOXVWUDWLQJWKDWVDPSOLQJRIQRGXOHV :KHWKHURUQRWWKHVH¿QGLQJVDUHDSSOLFDEOHRQ DODUJHUVFDOHUHPDLQVHOXVLYHDVZHZHUHOLPLWHG ZDVDGHTXDWHWRUHSUHVHQWWUXHJHQRW\SLFULFKQHVV WRDVLQJOHORFDWLRQLQZHVWHUQ2UHJRQ6WLOOWR 0RUHWKDQRIVHTXHQFHVGHULYHGIURPERWK WKHEHVWRIRXUNQRZOHGJHWKLVLVWKH¿UVWVWXG\ Alnus host species clustered into one dominant FRPSDULQJKRVWFrankia associations of these JHQRW\SH '-%) ZKLFKZDVDVVRFLDWHGZLWK species at a natural site. WUHHVWKDWZHUHKHWHURJHQHRXVO\GLVWULEXWHGDFURVV 6LPLODUWRRWKHUVWXGLHVZHIRXQGORZRYHUDOO WKHVLWH )LJXUH 6LPLODUO\WUHHVDVVRFLDWHGZLWK GLYHUVLW\RIFrankia %HQVRQHWDO&ODZVRQ WKHRWKHUIRXUJHQRW\SHV '-%)±'-%) DOVR HWDO2DNOH\HWDO9DQGHQ+HXYHOHW H[KLELWHGKHWHURJHQHRXVVSDWLDOGLVWULEXWLRQ8SRQ DO/LSXVDQG.HQQHG\3RNKDUHOHWDO comparing the Alnus Frankia DVVRFLDWHG JHQRW\SHV ZLWKDVLQJOHGRPLQDQWJHQRW\SHDFURVVWKH LQRXUVWXG\WRWKRVHFKDUDFWHUL]HGLQRWKHUVWXGLHV VDPSOLQJVLWH,QWHUHVWLQJO\WZRRIWKHJHQRW\SHV FRQGXFWHGLQRXUUHJLRQ .HQQHG\HWDO UHFRYHUHGLQWKHFXUUHQWVWXG\ZHUHLGHQWLFDOWRWZR /LSXVDQG.HQQHG\ ZHGLVFRYHUHGWKDW GHVFULEHGLQSUHYLRXVVWXGLHV 7DEOH WKDWZHUH WZRRIRXUJHQRW\SHV '-%)DQG'-%) ZHUH IRXQGDWVLWHVEHWZHHQNPDQGNPIURP LGHQWLFDOWRWZRGHVFULEHGSUHYLRXVO\ ./DQG WKHFXUUHQWVWXG\VLWH5HPDUNDEO\WKHVHPDWFK ./UHVSHFWLYHO\ 7KHUHVXOWVRIWKH)LVKHU¶V LQJJHQRW\SHVFRUUHVSRQGWRWKHPRVWDEXQGDQW ([DFW3UREDELOLW\7HVW P LQGLFDWHWKDW JHQRW\SHLQRXUVWXG\DQGWKHWZRPRVWDEXQGDQW QRV\VWHPDWLFDVVRFLDWLRQH[LVWVEHWZHHQAlnus LQWKRVHRI.HQQHG\HWDO DQG/LSXVDQG species and occurrence of Frankia JHQRW\SHLQ .HQQHG\ IXUWKHUXQGHUVFRULQJWKHUHJLRQDO at this site. ORZGLYHUVLW\RIAlnusDVVRFLDWHGFrankia. There ZHUHQRJHQRW\SHVWKDWZHUHXQLTXHWRHLWKHUKRVW Phylogenetic Analysis DQGQRVLJQL¿FDQWDVVRFLDWLRQEHWZHHQWKHDEXQ GDQFHRIJHQRW\SHVDQGKRVWVSHFLHVZDVREVHUYHG %RWKPD[LPXPOLNHOLKRRGDQG%D\HVLDQSK\OR LQGLFDWLQJDODFNRIKRVWVSHFL¿FLW\LQWKLVV\VWHP JHQHWLFDQDO\VHVRIWKH¿YHFrankia JHQRW\SHV $VLPLODUSDWWHUQZDVREVHUYHGE\3RNKDUHOHW UHFRYHUHGIURPA. rubra and A. rhombifolia DO ZKRIRXQGWKDW5HS3&5SUR¿OHV LH \LHOGHGWUHHVZLWKLGHQWLFDOWRSRORJLHV )LJXUH JHQRW\SHV RIFrankia associated with nine out of 7KHJHQRW\SHVDUHFOXVWHUHGLQWRWZRPDMRU Alnus WD[DLQDFRPPRQJDUGHQVHWWLQJZHUH FODGHVZLWKRQHFODGHFRQVLVWLQJRI'-%)DQG LGHQWLFDO/LNHZLVH2DNOH\HWDO SHUIRUPHG WKHRWKHUFRQVLVWLQJRIWZRPRQRSK\OHWLFVXE DVHTXHQFHEDVHGDQDO\VLVRIFrankia associated clades containing two of each of the remaining ZLWKVHYHUDOFRRFFXUULQJCeanothus species and IRXUJHQRW\SHV '-%)'-%)DQG'-%) UHSRUWHGDKLJKOHYHORIVHTXHQFHKRPRJHQHLW\ '-%)UHVSHFWLYHO\ 5HODWLYHO\KLJKERRWVWUDS DFURVVKRVWVGHVSLWHQRWKDYLQJFOXVWHUHGVLPLODU VXSSRUWYDOXHVDQGSRVWHULRUSUREDELOLWLHVVXSSRUW VHTXHQFHVLQWR278VZKLFKZRXOGKDYHOLNHO\ WKHVWDELOLW\RIWKHVHJURXSLQJV IXUWKHUUHGXFHGWKHREVHUYHGGLYHUVLW\,QWHUHVW

Balkan et al.

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Diversity of Frankia in Alnus

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Diversity of Frankia in Alnus

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Balkan et al.

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