Cryphonectriaceae associated with rust-infected Syzygium jambos in Hawaii.

Syzygium jambos (Myrtales, Myrtaceae) trees in Hawaii are severely affected by a rust disease caused by Austropuccinia psidii (Pucciniales, Sphaerophragmiaceae), but they are commonly co-infected with species of Cryphonectriaceae (Diaporthales). In this study, S. jambos and other trees in the Myrtales were examined on three Hawaiian Islands for the presence of Cryphonectriaceae. Bark samples with fruiting bodies were collected from infected trees and fungi were isolated directly from these structures. Pure cultures were produced and the fungi were identified using DNA sequence data for the internal transcribed spacer (ITS) region, part of the ß-tubulin (BT1) gene and the transcription elongation factor-1α (TEF1) gene. Five species in three genera of Cryphonectriaceae were identified from Myrtaceae tree samples. These included Chrysoporthe deuterocubensis, Microthia havanensis and three previously-unknown taxa described here as Celoporthe hauoliensis sp. nov., Cel. hawaiiensis sp. nov. and Cel. paradisiaca sp. nov. Representative isolates of Cel. hauoliensis, Cel. hawaiiensis, Cel. paradisiaca, Chr. deuterocubensis and Mic. havanensis were used in artificial inoculation studies to consider their pathogenicity on S. jambos. Celoporthe hawaiiensis, Cel. paradisiaca and Chr. deuterocubensis produced lesions on young S. jambos trees in inoculation trials, suggesting that, together with A. psidii, they may contribute to the death of trees. Microsatellite markers were subsequently used to consider the diversity of Chr. deuterocubensis on the Islands and thus to gain insights into its possible origin in Hawaii. Isolates of this important Myrtaceae and particularly Eucalyptus pathogen were found to be clonal. This provides evidence that Chr. deuterocubensis was introduced to the Hawaiian Islands as a single introduction, from a currently unknown source.

Characterization of Ti Ringspot-Associated Virus, a Novel Emaravirus Associated with an Emerging Ringspot Disease of Cordyline fruticosa.

Ti ringspot is an emerging foliar disease of the ti plant (Cordyline fruticosa) in Hawaii that is quickly spreading throughout the islands. Symptoms include small chlorotic ringspots on leaves that often coalesce to form larger lesions. Although several virus species have been discovered in symptomatic plants, none have been associated with these symptoms. Here, we report and characterize a novel virus closely associated with ti ringspot symptoms in Hawaii. The presence of double membrane bodies approximately 85 nm in diameter in symptomatic cells and sequence analyses of five genomic RNA segments obtained by high-throughput sequencing indicate that this virus is most closely related to members of the plant virus genus Emaravirus. Phylogenetic and sequence homology analyses place this virus on a distinct clade within the Emaravirus genus along with High Plains wheat mosaic emaravirus, blue palo verde broom virus, and Raspberry leaf blotch emaravirus. Sequence identity values with taxonomically relevant proteins indicate that this represents a new virus species, which we are tentatively naming ti ringspot-associated virus (TiRSaV). TiRSaV-specific reverse transcription PCR assays detected the virus in several experimental herbaceous host species following mechanical inoculation. TiRSaV was also detected in eriophyid mites collected from symptomatic ti plants, which may represent a putative arthropod vector of the virus.

Fusarium species as pathogen on orchids.

The recent surge in demand for exotic ornamental crops such as orchids has led to a rise in international production, and a sharp increase in the number of plant and plant products moving between countries. Along with the plants, diseases are also being transported and introduced into new areas. Fusarium is one of the major diseases causing pathogens infecting orchids that is spreading through international trade. Studies have identified several species of Fusarium associated with orchids, some are pathogenic and cause symptoms such as leaf and flower spots, leaf or sheath blights, pseudostem or root rots, and wilts. Infection and damage caused by Fusarium reduces the quality of plants and flowers, and can cause severe economic losses. This review documents the current status of the Fusarium-orchid interaction, and illustrates challenges and future perspectives based on the available literature. This review is the first of Fusarium and orchid interactions, and integrates diverse results that both furthers the understanding and knowledge of this disease complex, and will enable the development of effective disease management practices.

A Phytophthora palmivora Extracellular Cystatin-Like Protease Inhibitor Targets Papain to Contribute to Virulence on Papaya.

Papaya fruits, stems, and leaves are rich in papain, a cysteine protease that has been shown to mediate plant defense against pathogens and insects. Yet the oomycete Phytophthora palmivora is a destructive pathogen that infects all parts of papaya plants, suggesting that it has evolved cysteine protease inhibitors to inhibit papain to enable successful infection. Out of five putative extracellular cystatin-like cysteine protease inhibitors (PpalEPICs) from P. palmivora transcriptomic sequence data, PpalEPIC8 appeared to be unique to P. palmivora and was highly induced during infection of papaya. Purified recombinant PpalEPIC8 strongly inhibited papain enzyme activity, suggesting that it is a functional cysteine protease inhibitor. Homozygous PpalEPIC8 mutants were generated using CRISPR/Cas9-mediated gene editing via Agrobacterium-mediated transformation (AMT). Increased papain sensitivity of in-vitro growth and reduced pathogenicity during infection of papaya fruits were observed for the mutants compared with the wild-type strain, suggesting that PpalEPIC8, indeed, plays a role in P. palmivora virulence by inhibiting papain. This study provided genetic evidence demonstrating that plant-pathogenic oomycetes secrete cystatins as important weapons to invade plants. It also established an effective gene-editing system for P. palmivora by the combined use of CRISPR/Cas9 and AMT, which is expected to be applicable to other oomycetes.

Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora.

BACKGROUND: As an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility. RESULTS: In this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion. CONCLUSIONS: This system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.

Differentiation and distribution of cordyline viruses 1-4 in Hawaiian ti plants (Cordyline fruticosa L.).

Common green ti plants (Cordyline fruticosa L.) in Hawaii can be infected by four recently characterized closteroviruses that are tentative members of the proposed genus Velarivirus. In this study, a reverse-transcription polymerase chain reaction (RT-PCR) assay developed to detect and distinguish Cordyline virus 1 (CoV-1), CoV-2, CoV-3, and CoV-4 was used to determine: (i) the distribution of these viruses in Hawaii; and (ii) if they are involved in the etiology of ti ringspot disease. One hundred and thirty-seven common green ti plants with and without ti ringspot symptoms were sampled from 43 sites on five of the Hawaiian Islands and underwent the RT-PCR assay. Eleven ornamental ti varieties were also sampled and assayed. Based on this survey, it appears none of the CoVs are involved in the etiology of ti ringspot. The observation of a non-uniform geographic distribution of the CoVs in common green ti, combined with the presence of CoVs in seed-derived ornamental varieties, suggests active vector transmission. Eight herbarium specimens collected between 1903 and 2003 from plants on the island of Oahu also underwent the RT-PCR assay. Amplifiable RNA was isolated from accessions collected in 1985 or later, however only the 2003 accession was found to harbor CoVs.

Molecular characterization of closteroviruses infecting Cordyline fruticosa L. in Hawaii.

In Hawaii, common green ti plants (Cordyline fruticosa L.) have been shown to harbor Cordyline virus 1 (CoV-1) which, along with Little cherry virus 1 (LChV-1), and Grapevine leafroll-associated virus 7 (GLRaV-7), form a distinct clade within the family Closteroviridae. Preliminary work has indicated that, aside from CoV-1, three additional closteroviruses may infect common green ti plants in Hawaii. In this study, pyrosequencing was used to characterize the genomes of closteroviruses infecting a single common green ti plant. The sequence data confirmed the presence of CoV-1 as well as three additional closteroviruses. Although all four viruses had the same general genome organization, the sequence divergence between the RNA-dependent RNA polymerase, heat shock protein 70 homolog, and coat protein ranged from 22 to 61%, indicating these represent four distinct closterovirus species. The names CoV-2, CoV-3, and CoV-4 are proposed for the three new viruses. Phylogenetic analyses placed CoV-2, CoV-3, and CoV-4 in the same clade as CoV-1, LChV-1, and GLRaV-7.

AFLP, Pathogenicity, and VCG Analyses of Fusarium oxysporum and Fusarium pseudocircinatum from Acacia koa.

Acacia koa (koa), a native tree in Hawaii, suffers from a dieback caused by Fusarium oxysporum. Pathogenicity tests, vegetative compatibility group (VCG) tests, and amplified fragment length polymorphisms (AFLP) analyses were conducted on Fusarium isolates recovered from diseased koa. Koa seedling mortality with individual strains ranged from 0 to 85%, with 42% of the strains killing no seedlings. Thus, strains of F. oxysporum recovered from dying koa trees may or may not be virulent. In addition to F. oxysporum, F. pseudocircinatum strains were isolated from diseased koa; however, they were either nonvirulent or had weak virulence. This is the first report of F. pseudocircinatum in Hawaii. The 46 strains of F. oxysporum and F. pseudocircinatum were grouped into 16 VCGs, but 86% of the highly virulent strains belonged to VCG 2. In AFLP analyses, strains from the same VCG generally clustered with one another. Identification of the same set of strains using VCG, AFLP, and pathogenicity tests showed that the highly virulent strains are genetically close and that high virulence toward koa is not a property of all strains of F. oxysporum. Thus, VCG 2 with the corresponding AFLP data is a significant biological entity for which we propose the name F. oxysporum f. sp. acaciae to reflect its virulence on koa.

Pathogenicity, Internal Transcribed Spacer-rDNA Variation, and Human Dispersal of Ceratocystis fimbriata on the Family Araceae.

ABSTRACT Ceratocystis fimbriata is a complex of many cryptic, host-specialized species that causes wilt and canker of woody species and rot diseases of storage roots and corms of many economically important plants worldwide. With the exception of the family Araceae, all confirmed hosts of C. fimbriata are dicotyledonous plants. We hypothesized that the isolates from members of the family Araceae would form a monophyletic lineage specialized to infect these unique hosts. Analyses of sequences of the internal transcribed spacer (ITS) region of nuclear rDNA indicate that isolates and herbarium specimens of C. fimbriata from the family Araceae represent three different groups: an Xanthosoma/Syngonium group on corms of Xanthosoma spp. from the Caribbean region and on ornamental S. podophyllum from greenhouses in Florida, Hawaii, Australia, and Brazil; an inhame group on corms of Colocasia esculenta in Brazil; and a distantly related taro group on Colocasia esculenta in Hawaii and China and on X. sagittifolium in Fiji. Inoculations of three species of Araceae (Caladium bicolor, S. podophyllum, and Colocasia esculenta) showed that isolates from all three groups are pathogenic to these three hosts. Brazilian isolates from Mangifera indica and Ficus carica were only weakly pathogenic to Caladium and Syngonium sp. and were not pathogenic to Colocasia sp. Syngonium plants appeared to be most susceptible to isolates of the Xanthosoma/Syngonium group, and Colocasia plants were least susceptible to isolates from Syngonium spp. Thus, it appears that adaptations to the family Araceae have evolved more than once in the C. fimbriata complex. It is hypothesized that the three groups of C. fimbriata on the family Araceae are native to the Caribbean, Brazil, and Asia, respectively, but they have been spread elsewhere by humans.