Co-occurrence and genotypic distribution of Phytophthora species recovered from watersheds and plant nurseries of eastern Tennessee

In 2008 statewide surveys of symptomatic foliage of nursery plants from Tennessee resulted in isolation of 43 isolates of Phytophthora spp. This sample set includes four described species (P. citrophthora, P. citricola, P. nicotianae, P. syringae), and a provisional species of Phytophthora ('P. hydropathica'). At the same time a stream-baiting survey was initiated to recover Phytophthora from eight watersheds in eastern Tennessee, some of which are near plant nurseries. Baiting was accomplished by submerging healthy Rhododendron leaves approximately 1 wk and isolation onto selective media. Six baiting periods were completed, and in total 98 Phytophthora isolates and 45 isolates of Pythium spp. were recovered. Three described species (P. citrophthora, P. citricola and P. irrigata) and the provisional species 'P. hydropathica' were obtained as well as three undescribed Phytophthora taxa and Pythium litorale. Isolates from both surveys were identified to species with morphology and the internal transcribed spacer (ITS) sequence. Isolates from species co-occurring in streams and nurseries (P. citricola, P. citrophthora and 'P. hydropathica') were characterized further with amplified fragment length polymorphism (AFLP) analyses and mefenoxam tolerance assays. Isolates representing a putative clonal genotype of P. citricola were obtained from both environmental and nursery sample sets.     

Targeted gene mutation in Phytophthora spp

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosisinducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.     

A strategy for recovering high quality genomic DNA from a large number of Phytophthora isolates

We present a strategy to recover high molecular weight genomic DNA from large numbers of isolates of Phytophthora. Included are steps for generating mycelial mass in 24-well reuseable deep well plates, efficient lyophilization and disruption of the mycelium and genomic DNA extraction with 96-well glass fiber filter plates. The resulting DNA is consistently high molecular weight and is suitable for applications that require high quality DNA such as AFLP analysis and TILLING. A single operator easily can manage mycelium preparation and/or DNA extraction from 384 isolates in a single day and this approach might be useful for other fungi or fungi-like organisms that can be grown in liquid media.     

Natural Infection of Field-Grown Daikon Radish (Raphanus sativus L.) Crops by Turnip Crinkle Virus in North America

In 1993, stunted field-grown daikon radish plants were found with black lesions on the petioles and leaf blades, and new growth exhibited leaf crinkling and a mosaic pattern on distorted leaves. The plants had hollow roots and seed production was severely reduced. Symptomatic plants contained isometric virions identified as those of turnip crinkle virus (TCV). This is the first report of TCV causing serious economic damage and the first report of natural infection in North America.