Detection and Molecular Phylogenetic-Morphometric Characterization of Rhizoctonia tuliparum, Causal Agent of Gray Bulb Rot of Tulips and Bulbous Iris.

Gray bulb rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen, Rhizoctonia tuliparum (Rtul). Sclerotia present in infected bulbs, as well as overwintering sclerotia in soil and field debris, are the primary sources of infection. A method for accurate and sensitive detection of Rtul from soil and infected bulbs, and estimation of inoculum threshold levels, is needed for the management of disease caused by this pathogen. We designed a unique set of primers targeting the ITS2 region of the Rtul genome and developed a highly sensitive quantitative PCR (qPCR)-based method for Rtul identification using these primers, where the threshold of detection was approximately 1 fg Rtul DNA. The assay was more sensitive with sclerotia collected from the field (natural) than with those grown in the lab, and more sensitive with natural-light than natural-dark sclerotia. Also, the detection method was more sensitive when sclerotia were extracted from soil than from bulb tissue. The qPCR method was highly specific, as no PCR amplification was detected when genomic DNA from 62 non-Rtul Rhizoctonia isolates from a wide range of anastomosis groups were tested. To understand the evolutionary relationships and genomic diversity of Rtul, we performed phylogenetics of the ITS1-5.8S-ITS2 region and ITS2-molecular morphometric characterization (MMC) of Rtul isolates. The three Rtul isolates whose ITS sequences were available in GenBank formed a distinct phylogenetic clade with Ceratobasidium anceps as the nearest relative. Furthermore, MMC analysis revealed genetic divergence among these three Rtul isolates.

Genetic analysis reveals unprecedented diversity of a globally-important plant pathogenic genus.

Genus Botrytis contains approximately 35 species, many of which are economically-important and globally-distributed plant pathogens which collectively infect over 1,400 plant species. Recent efforts to genetically characterize genus Botrytis have revealed new species on diverse host crops around the world. In this study, surveys and subsequent genetic analysis of the glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and necrosis and ethylene-inducing proteins 1 and 2 (NEP1 and NEP2) genes indicated that Botrytis isolates collected from peony fields in the United States contained more species diversity than ever before reported on a single host, including up to 10 potentially novel species. Together, up to 16 different phylogenetic species were found in association with peonies in the Pacific Northwest, which is over a third of the total number of species that are currently named. Furthermore, species were found on peonies in Alaska that have been described on other host plants in different parts of the world, indicating a wider geographic and host distribution than previously thought. Lastly, some isolates found on peony share sequence similarity with unnamed species found living as endophytes in weedy hosts, suggesting that the isolates found on peony have flexible lifestyles as recently discovered in the genus. Selected pathogenicity, growth, and morphological characteristics of the putatively new Botrytis species were also assessed to provide a basis for future formal description of the isolates as new species.

Characterization of phenotypic variation and genome aberrations observed among Phytophthora ramorum isolates from diverse hosts.

BACKGROUND: Accumulating evidence suggests that genome plasticity allows filamentous plant pathogens to adapt to changing environments. Recently, the generalist plant pathogen Phytophthora ramorum has been documented to undergo irreversible phenotypic alterations accompanied by chromosomal aberrations when infecting trunks of mature oak trees (genus Quercus). In contrast, genomes and phenotypes of the pathogen derived from the foliage of California bay (Umbellularia californica) are usually stable. We define this phenomenon as host-induced phenotypic diversification (HIPD). P. ramorum also causes a severe foliar blight in some ornamental plants such as Rhododendron spp. and Viburnum spp., and isolates from these hosts occasionally show phenotypes resembling those from oak trunks that carry chromosomal aberrations. The aim of this study was to investigate variations in phenotypes and genomes of P. ramorum isolates from non-oak hosts and substrates to determine whether HIPD changes may be equivalent to those among isolates from oaks. RESULTS: We analyzed genomes of diverse non-oak isolates including those taken from foliage of Rhododendron and other ornamental plants, as well as from natural host species, soil, and water. Isolates recovered from artificially inoculated oak logs were also examined. We identified diverse chromosomal aberrations including copy neutral loss of heterozygosity (cnLOH) and aneuploidy in isolates from non-oak hosts. Most identified aberrations in non-oak hosts were also common among oak isolates; however, trisomy, a frequent type of chromosomal aberration in oak isolates was not observed in isolates from Rhododendron. CONCLUSION: This work cross-examined phenotypic variation and chromosomal aberrations in P. ramorum isolates from oak and non-oak hosts and substrates. The results suggest that HIPD comparable to that occurring in oak hosts occurs in non-oak environments such as in Rhododendron leaves. Rhododendron leaves are more easily available than mature oak stems and thus can potentially serve as a model host for the investigation of HIPD, the newly described plant-pathogen interaction.

Comparison of Five Detection and Quantification Methods for Phytophthora ramorum in Stream and Irrigation Water.

Propagules of Phytophthora ramorum, the causal agent of sudden oak death (SOD) and ramorum blight, can be recovered from infested stream and nursery irrigation runoff using baiting and filtration methods. Five detection methods, including pear and rhododendron leaf baits, Bottle O' Bait, filtration, and quantitative polymerase chain reaction (qPCR) performed on zoospores trapped on a filter were compared simultaneously in laboratory assays using lab or creek water spiked with known quantities of P. ramorum zoospores. The detection threshold for each method was determined and methods that could be used to quantify zoospore inoculum were identified. Filtration and qPCR were the most sensitive at detecting low levels of zoospores, followed by wounded rhododendron leaves, rhododendron leaf disks, and pear baits. Filtration, qPCR, and leaf disks were able to quantify P. ramorum zoospores ranging from 2 to 451 direct-plate CFU/liter while wounded leaves and pear baits appeared to be better at detection rather than quantification. The ability to detect and quantify P. ramorum inoculum in water will assist scientists, regulatory agencies, and nursery personnel in assessing the risk of spreading P. ramorum in nurseries and landscape sites where untreated infested water is used for irrigation.