Analysis of Claviceps africana and C. sorghi from India using AFLPs, EF-1alpha gene intron 4, and beta-tubulin gene intron 3.

Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1alpha gene intron 4 and beta-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1alpha gene intron 4, the beta-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.

Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions.

ABSTRACT A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10(-5) dilution of extracted tissue of artificially infected rhododendron 'Cunningham's White', and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.

AFLP Comparisons Among Claviceps africana Isolates from the United States, Mexico, Africa, Australia, India, and Japan.

Eighty-seven isolates of the sorghum ergot pathogen, Claviceps africana, from diverse geographic locations were analyzed using four different amplified fragment length polymorphism (AFLP) primer combinations to determine genetic relationships among isolates. Most isolates showed unique AFLP haplotypes, indicating that substantial genetic variation is present within C. africana populations. Two major groupings of isolates were observable, with ca. 70% similarity between the two groups. One group consisted of Australian, Indian, and Japanese isolates and the other of U.S., Mexican, and African isolates. In spite of overall high levels of genetic diversity observed in C. africana, isolates within the two major groups were between 75 and 100% similar. The observed associations of C. africana isolates from worldwide sources could be the result of intercontinental trade and/or movement of seed. The data indicate that Africa was the likely source of C. africana that has become established in the Americas since 1996. Analysis of additional isolates in future studies will reveal whether these groupings are being maintained or whether population subdivision or reshuffling may occur.