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A New Subclade of Leptosphaeria biglobosa Identified from Brassica rapa.

ABSTRACT: Blackleg (Phoma stem canker) of crucifers is a globally important disease caused by the ascomycete species complex comprising of Leptosphaeria maculans and Leptosphaeria biglobosa. Six blackleg isolates recovered from Brassica rapa cv. Mizspoona in the Willamette Valley of Oregon were characterized as L. biglobosa based on standard pathogenicity tests and molecular phylogenetic analysis. These isolates were compared to 88 characterized L. biglobosa isolates from western Canada, 22 isolates from Australia, and 6 L. maculans isolates from Idaho, USA using maximum parsimony and distance analysis of phylogenetic trees generated from the ITS rDNA (internal transcribed spacer rDNA) sequence, and the actin and ?-tubulin gene sequences. The L. biglobosa isolates derived from B. rapa collected in Oregon formed a separate subclade based on concatenated gene sequences or a single gene sequence, regardless of the analyses. Pathogenicity tests showed that these isolates failed to infect either resistant or susceptible B. napus cultivars, but caused severe symptoms on three B. rapa cultivars (Accession number: UM1113, UM1112, and UM1161), a B. oleracea var. capitata (cabbage) cultivar (Copenhagen Market), and two B. juncea cultivars (CBM, a common brown Mustard, and Forge). These findings demonstrated that the L. biglobosa isolates derived from a B. rapa crop in Oregon were genetically distinct from existing species of L. biglobosa, and constitute a new subclade, herein proposed as L. biglobosa 'americensis'.

SUBMITTER: Zou Z

PROVIDER: S-EPMC6479289 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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A New Subclade of Leptosphaeria biglobosa Identified from Brassica rapa.

Zou Zhongwei Z Zhang Xuehua X Parks Paula P du Toit Lindsey J LJ Van de Wouw Angela P AP Fernando W G Dilantha WGD

International journal of molecular sciences 20190403 7

Blackleg (Phoma stem canker) of crucifers is a globally important disease caused by the ascomycete species complex comprising of Leptosphaeria maculans and Leptosphaeria biglobosa. Six blackleg isolates recovered from Brassica rapa cv. Mizspoona in the Willamette Valley of Oregon were characterized as L. biglobosa based on standard pathogenicity tests and molecular phylogenetic analysis. These isolates were compared to 88 characterized L. biglobosa isolates fro ...[more]

PMID: 30987176

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Project description:Two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines, Chiifu (smaple A) and Kenshin (Smaple B), were grown for approximately 4 weeks in a growth chamber at 22degreesC under a 16 h light/8 h dark photoperiod with a photon flux density of 140 umol m-2 s-1. For microarray analysis, plants were exposed to a temperature of -4degreesC for 4 h, and shoots were sampled. Total RNA was isolated from the samples using an Easy-BLUETM Total RNA Extraction Kit (Invitrogen, NY, U.S.A.) and was then purified using an RNeasy MinEluteTM Cleanup Kit (Qiagen, Germany). Mircroarray experiments were carried out using Brassica 24K Oligo Microarray. RNA were prepared from each plant sample and 10 ug of total RNA were used for cDNA synthesis by using Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, NY, U.S.A.). Following cDNA synthesis, remaining RNA was removed by the addition of RNaseA and the cDNA was precipitated following phenol/chloroform extraction. The cDNA pellet was rehydrated and used for Cy3-labelling. For the synthesis of Cy3-labeled target DNA fragments, 1 ug of double strand cDNA was mixed with 40 ul (1 OD) of Cy3-9 mer primers (Sigma-Aldrich) and the total volume was adjusted to 80 ul with deionized water. After heating at 98degreesC for 10 min, 10 uL of dNTP mix (10 mM each) and 2 ul of Klenow fragment (NEB; 50 units/ul) were added and the reaction was incubated at 37degreesC for 2 h. Finally, the reaction was stopped by the addition of 10 ul of 0.5 M EDTA. Labeled DNA fragments were precipitated with isopropanol and rehydrated with water. The concentration of each sample was determined using spectrophotometer. For hybridization, 13 ug of the labeled DNA fragment was mixed with hybridization buffer (NimbleGen) and then hybridized with the microarray using a MAUI hybridization chamber (Biomicro) at 42degreesC for 16 h. Following hybridization, the microarray was washed with wash solution I, II, and III (NimbleGen), and then dried in a dark desiccator. The microarray slide was scanned using GenePix scanner 4000B (Axon) and the spot intensities were analyzed using a NimbleScan (NimbleGen). The normal distribution of Cy3 intensities was tested by qqline. The data was normalized and processed with cubic spline normalization using quantiles to adjust signal variations between chips and with Rubust Multi-Chip Analysis (RMA) using a median polish algorithm implemented in NimbleScan software (NimbleGen). Two biological replicates were carried out.

Dataset Information

A New Subclade of Leptosphaeria biglobosa Identified from Brassica rapa.

Publications

A New Subclade of <i>Leptosphaeria biglobosa</i> Identified from <i>Brassica rapa</i>.

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