Integration of early disease-resistance phenotyping, histological characterization, and transcriptome sequencing reveals insights into downy mildew resistance in impatiens.

Downy mildew (DM), caused by obligate parasitic oomycetes, is a destructive disease for a wide range of crops worldwide. Recent outbreaks of impatiens downy mildew (IDM) in many countries have caused huge economic losses. A system to reveal plant-pathogen interactions in the early stage of infection and quickly assess resistance/susceptibility of plants to DM is desired. In this study, we established an early and rapid system to achieve these goals using impatiens as a model. Thirty-two cultivars of Impatiens walleriana and I. hawkeri were evaluated for their responses to IDM at cotyledon, first/second pair of true leaf, and mature plant stages. All I. walleriana cultivars were highly susceptible to IDM. While all I. hawkeri cultivars were resistant to IDM starting at the first true leaf stage, many (14/16) were susceptible to IDM at the cotyledon stage. Two cultivars showed resistance even at the cotyledon stage. Histological characterization showed that the resistance mechanism of the I. hawkeri cultivars resembles that in grapevine and type II resistance in sunflower. By integrating full-length transcriptome sequencing (Iso-Seq) and RNA-Seq, we constructed the first reference transcriptome for Impatiens comprised of 48,758 sequences with an N50 length of 2060 bp. Comparative transcriptome and qRT-PCR analyses revealed strong candidate genes for IDM resistance, including three resistance genes orthologous to the sunflower gene RGC203, a potential candidate associated with DM resistance. Our approach of integrating early disease-resistance phenotyping, histological characterization, and transcriptome analysis lay a solid foundation to improve DM resistance in impatiens and may provide a model for other crops.

Characterization of Phytophthora spp. Isolated from Ornamental Plants in Florida.

This report investigates population structure and genetic variability of Phytophthora spp. isolated from botanically diverse plants in Florida. Internal transcribed spacer-based molecular phylogenetic analyses indicate that Phytophthora isolates recovered from ornamental plants in Florida represent a genetically diverse population and that a majority of the isolates belong to Phytophthora nicotianae (73.2%), P. palmivora (18.7%), P. tropicalis (4.9%), P. katsurae (2.4%), and P. cinnamomi (0.8%). Mating type analyses revealed that most isolates were heterothallic, consisting of both mating type A1 (25.2%) and mating type A2 (39.0%), and suggesting that they could outcross. Fungicide sensitivity assays determined that several isolates were moderate to completely insensitive to mefenoxam. In addition, several isolates were also moderately insensitive to additional fungicides with different modes of action. However, correlation analyses did not reveal occurrence of fungicide cross-resistance. These studies suggest that a genetically diverse Phytophthora population infects ornamental crops and the occurrence of mefenoxam-insensitive Phytophthora populations raises concerns about disease management in ornamentals. Mitigating fungicide resistance will require prudent management strategies, including tank mixes and rotation of chemicals with different modes of actions.

Development of Multilocus PCR Assays for Raffaelea lauricola, Causal Agent of Laurel Wilt Disease.

Laurel wilt, caused by the fungus Raffaelea lauricola, is an exotic disease that affects members of the Lauraceae plant family in the southeastern United States. The disease is spreading rapidly in native forests and is now found in commercial avocado groves in south Florida, where an accurate diagnostic method would improve disease management. A polymerase chain reaction (PCR) method based on amplifying the ribosomal small-subunit DNA, with a detection limit of 0.0001 ng, was found to be suitable for some quantitative PCR applications; however, it was not taxon specific. Genomic sequencing of R. lauricola was used to identify and develop primers to amplify two taxon-specific simple-sequence repeat (SSR) loci, which did not amplify from related taxa or host DNA. The new SSR loci PCR assay has a detection limit of 0.1 ng of R. lauricola DNA, is compatible with traditional and real-time PCR, was tested in four labs to confirm consistency, and reduces diagnostic time from 1 week to 1 day. Our work illustrates pitfalls to designing taxon-specific assays for new pathogens and that undescribed fungi can limit specificity.

A Comparison of Standard and High-Fidelity PCR: Evaluating Quantification and Detection of Pathogen DNA in the Presence of Orchid Host Tissue.

The polymerase chain reaction (PCR) has been used with increasing frequency for detecting and identifying plant pathogens. Although PCR is sensitive, research has shown that amplification of target microbial DNA from within another organism, such as an arthropod or plant, can be inhibited by the presence of host DNA. In this study, the sensitivity of standard and high-fidelity PCR, which incorporates a second DNA polymerase with proofreading ability, to detect and amplify DNA from the fungal pathogen Pseudocercospora odontoglossi while in the presence of Cattleya orchid DNA, was compared. Different dilutions of plasmids containing internal transcribed spacer (ITS)1, 5.8S, and ITS2 rDNA from P. odontoglossi were spiked with Cattleya orchid plant DNA. The high-fidelity PCR could detect and amplify as few as 207 plasmids containing the fungal DNA, whereas the standard PCR required over 200 million copies. The high-fidelity PCR was more efficient than conventional PCR in detecting Sclerotium rolfsii and a Dickeya sp. from freshly inoculated orchid plants, demonstrating its increased sensitivity in early detection of fungal and bacterial pathogens that are difficult to discriminate early in disease development.

Improved tolerance toward fungal diseases in transgenic Cavendish banana (Musa spp. AAA group) cv. Grand Nain.

The most devastating disease currently threatening to destroy the banana industry worldwide is undoubtedly Sigatoka Leaf spot disease caused by Mycosphaerella fijiensis. In this study, we developed a transformation system for banana and expressed the endochitinase gene ThEn-42 from Trichoderma harzianum together with the grape stilbene synthase (StSy) gene in transgenic banana plants under the control of the 35S promoter and the inducible PR-10 promoter, respectively. The superoxide dismutase gene Cu,Zn-SOD from tomato, under control of the ubiquitin promoter, was added to this cassette to improve scavenging of free radicals generated during fungal attack. A 4-year field trial demonstrated several transgenic banana lines with improved tolerance to Sigatoka. As the genes conferring Sigatoka tolerance may have a wide range of anti-fungal activities we also inoculated the regenerated banana plants with Botrytis cinerea. The best transgenic lines exhibiting Sigatoka tolerance were also found to have tolerance to B. cinerea in laboratory assays.

Influence of Temperature, Light Intensity, and Isolate on the Development of Neofusicoccum parvum-Induced Dieback of Eugenia, Syzygium paniculatum.

Syzygium paniculatum (Myrtaceae) is an important plant in the South Florida ornamental industry. Known as eugenia in the trade, the plant was relatively free of diseases before Hurricane Wilma (2005). Since then, a serious dieback disease has become prevalent in local nurseries, especially during late summer. Symptoms included wilting and death of terminal and lateral branches, and vascular discoloration in dead and dying branches and the main stem. Several fungi were isolated from diseased plants, but Neofusicoccum parvum was usually the only fungus isolated from symptomatic tissue. Most isolates were sterile, but all that were tested produced significant (P < 0.05) dieback on, and reduced growth of, the cultivar Monterrey Bay. Glomerella spp. and a Pestalotiopsis sp. that were recovered from asymptomatic portions of diseased plants and Mycoleptodiscus terrestris recovered from healthy liners of Monterrey Bay did not cause dieback symptoms in pathogenicity studies or affect host growth. In incubator studies, N. parvum caused significant external symptoms, vascular discoloration, and mortality at 25 and 30°C; in general, only vascular symptoms developed at 20°C and no symptoms developed at 15°C. Thus, temperature may be associated with the seasonal development of this disease. Significant differences in disease development were not observed under a wide range of light intensities (2,000 to 300 µmol s-1 m-2). S. paniculatum is a new host record for N. parvum.