Abstract
Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a ‘reporter’ gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.
Highlights
Coconut palm (Cocos nucifera L.) is one of the most economically important trees in the world
H2.1y.pShenaslitiinvoitcyuolfaCo. pfatrhaedowxailtdo -HtympBe strain X-3314 were inoculated on potato dextrose agar (PDA)
Hygromycin B (HmB) ranging from 10–40 μg/mL was used for testing the sensitivity of C. paradoxa to the antibiotic
Summary
Coconut palm (Cocos nucifera L.) is one of the most economically important trees in the world. Coconut stem-bleeding (CSB), caused by the fungus Ceratocystis paradoxa (De Seynes) Höhn (anamorph Thielaviopsis paradoxa), is one of the most notorious biotic diseases of coconut. In the state of Sergipe, Brazil, it was first detected in early 2004 [2] It was discovered in China for the first time in 2009, where it was found to occur in most coconut gardens [3]. The symptoms of CSB are described as follows: The affected trunk areas exhibit dark discoloration and a reddish-brown or rust-colored liquid bleeding from the stem cracks that may turn blackish when driedI,ntr.eJ.sMuoltl.iSncgi. 2i0n19r,o19t,tixng when moist; there is a reduced frequency of leaf emergence and a2 orfe1d1uced young leaf size, with stem-thinning occurring near the canopy as the disease progresses; and the leaves eventuallyThbeecsoympetbormoswonfisCh-SyBelalorewdaensdcrifbreadgilaes[2fo,3ll]o. Hcahsarbaceteenrisstuiccscaensdsfupalltyhoegxepnreessisseodf Cin. pnauramdoexraoaunsdaistscoinmteyraccettieosn, winicthlutdhienhgoCsto. lGlerteoetnricflhuuomresacceunttatum, Verticpilrloiuteminf(uGnFgPic)oelxapArecsrseimononhiausmprcohvreynsotospboeriauums,efaunldtoSool rfodrarsiumchaacnroaslpyosersa i[n14fi,l1a5m].enIntouthsifsusntguid[1y2,,t1h3e] first protoapnladsthapsrebpeaenrastiuocnceasnsfdulplyoleyxeptrheyssleendeinglnyucmole(rPoEusGa)-smcoemdyiacteeteds,triannclsufdoirnmgaCtioollnetsotyrsictheummfoacruCta.tpuamr,adoxa was eVsetratbicliilslihuemdf,uanngdicotlhaeAecxrepmroensisuimonchvreycstoospropriCumT,7a4n-sdGSFoPrdawriams atcrraonspsoforarm[14e,d15i]n. tIon tthhies fsutundgyu, sthteofoirbsttain a recompbrointoapnltasfut npgruepsalraabtieolnedawndithpGolFyePt.hOyluerneprgoltyoccooll (wPiElGl i)n-mfoerdmiarteedseatrracnhsftohramt aintivoonlvseysstmemonfiotor riCn.g the infectopiabortnaadipnoxroaa gwrreaecssosemisotbanibnolaifsnhtthefdius,nafgnuudnsgthluaebseeixlnepdtrhewsesiictohoncGvoeFncPut.otrOapnuCdrTps7hr4oo-tsuoGclFdoPlawwlsiaolsl itinrnaffonorsrmmfortmrheeseedfauirncrthtohttehhraeetfxuinpnvlgoourlsvaettoison of the mmecohnaitnoirsinmgsthoef iCn.fepcatrioandopxraogpraetshsioogneonficthitiys.fungus in the coconut and should inform the further exploration of the mechanisms of C. paradoxa pathogenicity
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