Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying Phytophthora Species

The genus Phytophthora is agriculturally and ecologically important. As the number of Phytophthora species continues to grow, identifying isolates in this genus has become increasingly challenging even by DNA sequencing. This study evaluated nine commonly used genetic markers against 154 formally described and 17 provisionally named Phytophthora species. These genetic markers were the cytochrome-c oxidase 1 (cox1), internal transcribed spacer region (ITS), 60S ribosomal protein L10, beta-tubulin (β-tub), elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA, and tigA gene fusion protein (tigA). As indicated by species distance, cox1 had the highest genus-wide resolution, followed by ITS, tigA, and β-tub. Resolution of these four markers also varied with (sub)clade. β-tub alone could readily identify all species in clade 1, cox1 for clade 2, and tigA for clades 7 and 8. Two or more genetic markers were required to identify species in other clades. For PCR consistency, ITS (99% PCR success rate) and β-tub (96%) were easier to amplify than cox1 (75%) and tigA (71%). Accordingly, it is recommended to take a two-step approach: classifying unknown Phytophthora isolates to clade by ITS sequences, as this marker is easy to amplify and its signature sequences are readily available, then identifying to species by one or more of the most informative markers for the respective (sub)clade

Zoospore interspecific signaling promotes plant infection by Phytophthora

<p>Abstract</p> <p>Background</p> <p>Oomycetes attack a huge variety of economically and ecologically important plants. These pathogens release, detect and respond to signal molecules to coordinate their communal behaviors including the infection process. When signal molecules are present at or above threshold level, single zoospores can infect plants. However, at the beginning of a growing season population densities of individual species are likely below those required to reach a quorum and produce threshold levels of signal molecules to trigger infection. It is unclear whether these molecules are shared among related species and what their chemistries are.</p> <p>Results</p> <p>Zoospore-free fluids (ZFF) from <it>Phytophthora capsici</it>, <it>P. hydropathica</it>, <it>P. nicotianae </it>(ZFFnic), <it>P. sojae </it>(ZFFsoj) and <it>Pythium aphanidermatum </it>were cross tested for stimulating plant infection in three pathosystems. All ZFFs tested significantly increased infection of <it>Catharanthus roseus </it>by <it>P. nicotianae</it>. Similar cross activities were observed in infection of <it>Lupinus polyphyllus </it>and <it>Glycine max </it>by <it>P. sojae</it>. Only ZFFnic and ZFFsoj cross induced zoospore aggregation at a density of 2 × 10³ml^-1. Pure autoinducer-2 (AI-2), a component in ZFF, caused zoospore lysis of <it>P. nicotianae </it>before encystment and did not stimulate plant infection at concentrations from 0.01 to 1000 μM. <it>P. capsici </it>transformants with a transiently silenced AI-2 synthase gene, ribose phosphate isomerase (<it>RPI</it>), infected <it>Capsicum annuum </it>seedlings at the same inoculum concentration as the wild type. Acyl-homoserine lactones (AHLs) were not detected in any ZFFs. After freeze-thaw treatments, ZFF remained active in promoting plant infection but not zoospore aggregation. Heat treatment by boiling for 5 min also did not affect the infection-stimulating property of ZFFnic.</p> <p>Conclusion</p> <p>Oomycetes produce and use different molecules to regulate zoospore aggregation and plant infection. We found that some of these signal molecules could act in an inter-specific manner, though signals for zoospore aggregation were somewhat restricted. This self-interested cooperation among related species gives individual pathogens of the same group a competitive advantage over pathogens and microbes from other groups for limited resources. These findings help to understand why these pathogens often are individually undetectable until severe disease epidemics have developed. The signal molecules for both zoospore aggregation and plant infection are distinct from AI-2 and AHL.</p

Endophytic Burkholderia sp. SSG as a potential biofertilizer promoting boxwood growth

Background Burkholderia sp. SSG is a bacterial endophyte isolated from boxwood leaves showing a resistant response to infection by the boxwood blight pathogen Calonectria pseudonaviculata. SSG acted as a protective and curative biocontrol agent for boxwood blight and as a bio-sanitizer of disease inoculum in the field. Many gene clusters involved in antibiotic production and plant growth promotion (PGP) were found in the genome, giving this endophyte great application potential as a treatment for plant protection. However, the PGP features have not been documented. This study investigated the plant growth promotion activity of SSG in boxwood. Methods To determine whether SSG is a plant growth promoting bacterium, four PGP traits, auxin and siderophore production, nitrogen fixation and phosphate solubilization, were examined in the laboratory with colorimetric or agar plate assays. The plant growth promoting activity of SSG was tested on three boxwood varieties characterized by slow, intermediate and fast growth rates, namely Justin Brouwers, Buddy and Winter Gem, respectively. These plants were drenched with an SSG cell suspension or water and washed plant weight was compared before and after treatment to determine growth changes after 10 months. Results The SSG culture was sustainable on nitrogen free media, suggesting that SSG may fix atmospheric nitrogen. It was also a strong phosphate solubilizer and a potent siderophore and indole-3-acetic acid (IAA) producer. Significant growth promotion was observed on boxwood cultivars Justin Brouwers, Buddy and Winter Gem 10 months after plant roots were drenched with SSG cells. The growth rate of treated plants was 76.1, 58.3, and 37.3% higher than that of the control, respectively. The degree of growth promotion was significantly different among plant varieties, notably more pronounced with the slow and intermediate growers. This study demonstrates that the SSG bacterium has multiple PGP traits and is a prospective plant biofertilizer

Phytophthora ×stagnum nothosp. nov., a new hybrid from irrigation reservoirs at ornamental plant nurseries in Virginia.

A novel Phytophthora species was frequently recovered from irrigation reservoirs at several ornamental plant production facilities in eastern Virginia. Initial sequencing of the internal transcribed spacer (ITS) region of this species generated unreadable sequences due to continual polymorphic positions. Cloning and sequencing the ITS region as well as sequencing the mitochondrially encoded cytochrome c oxidase 1 and beta-tubulin genes revealed that it is a hybrid between P. taxon PgChlamydo as its paternal parent and an unknown species genetically close to P. mississippiae as its maternal parent. This hybrid has some diagnostic morphological features of P. taxon PgChlamydo and P. mississippiae. It produces catenulate hyphal swellings, characteristic of P. mississippiae, and chlamydospores, typical of P. taxon PgChlamydo. It also produces both ornamented and relatively smooth-walled oogonia. Ornamented oogonia are another important diagnostic character of P. mississippiae. The relatively smooth-walled oogonia may be indicative of oogonial character of P. taxon PgChlamydo. The new hybrid is described here as Phytophthora ×stagnum

Potential Distribution of Invasive Boxwood Blight Pathogen (Calonectriapseudonaviculata) as Predicted by Process-Based and Correlative Models

Boxwood blight caused by Cps is an emerging disease that has had devastating impacts on Buxus spp. in the horticultural sector, landscapes, and native ecosystems. In this study, we produced a process-based climatic suitability model in the CLIMEX program and combined outputs of four different correlative modeling algorithms to generate an ensemble correlative model. All models were fit and validated using a presence record dataset comprised of Cps detections across its entire known invaded range. Evaluations of model performance provided validation of good model fit for all models. A consensus map of CLIMEX and ensemble correlative model predictions indicated that not-yet-invaded areas in eastern and southern Europe and in the southeastern, midwestern, and Pacific coast regions of North America are climatically suitable for Cps establishment. Most regions of the world where Buxus and its congeners are native are also at risk of establishment. These findings provide the first insights into Cps global invasion threat, suggesting that this invasive pathogen has the potential to significantly expand its range

Differential Adaptation Has Resulted in Aggressiveness Variation of Calonectria pseudonaviculata on Hosts Buxus, Pachysandra, and Sarcococca

Calonectria pseudonaviculata (Cps) infects Buxus (boxwood), Pachysandra (pachysandra), and Sarcococca spp. (sweet box); yet, how it adapts to its hosts has been unclear. Here, we performed serial passage experiments with the three hosts and measured Cps changes in three aggressiveness components: infectibility, lesion size, and conidial production. The detached leaves of individual hosts were inoculated with isolates (P0) from the originating host, followed by nine serial inoculations of new leaves of the same host with conidia from the infected leaves of the previous inoculation. All boxwood isolates maintained their capability of infection and lesion expansion through the 10 passages, whereas most non-boxwood isolates lost these abilities during the passages. Isolates from plants of origin (*-P0) and their descendants isolated from passages 5 (*-P5) and 10 (*-P10) were used to evaluate aggressiveness changes on all three hosts with cross-inoculation. While post-passage boxwood isolates gave enlarged lesions on pachysandra, sweet box P5 and pachysandra P10 isolates showed reduced aggressiveness on all hosts. Cps appears to be most adapted to boxwood and less adapted to sweet box and pachysandra. These results suggest speciation of Cps, with its coevolutionary pace with the hosts the fastest with boxwood, intermediate with sweet box, and the slowest with pachysandra

cDNA Transcriptome of <i>Arabidopsis</i> Reveals Various Defense Priming Induced by a Broad-Spectrum Biocontrol Agent <i>Burkholderia</i> sp. SSG

Burkholderia sp. SSG is a potent biological control agent. Even though its survival on the leaf surface declined rapidly, SSG provided extended, moderate plant protection from a broad spectrum of pathogens. This study used Arabidopsis Col-0 and its mutants, eds16-1, npr1-1, and pad4-1 as model plants and compared treated plants with non-treated controls to elucidate whether SSG triggers plant defense priming. Only eds16-1 leaves with SSG became purplish, suggesting the involvement of salicylic acid (SA) in SSG-induced priming. cDNA sequencing of Col-0 plants and differential gene expression analysis identified 120 and 119 differentially expressed genes (DEGs) at 6- and 24-h post-treatment (hpt) with SSG, respectively. Most of these DEGs encoded responses to biotic and abiotic stimuli or stresses; four DEGs had more than two isoforms. A total of 23 DEGs were shared at 6 and 24 hpt, showing four regulation patterns. Functional categorization of these shared DEGs, and 44 very significantly upregulated DEGs revealed that SSG triggered various defense priming mechanisms, including responses to phosphate or iron deficiency, modulation of defense-linked SA, jasmonic acid, ethylene, and abscisic acid pathways, defense-related gene regulation, and chromatin modification. These data support that SSG is an induced systemic resistance (ISR) trigger conferring plant protection upon pathogen encounter

Differential Tolerance to <i>Calonectria pseudonaviculata</i> of English Boxwood Plants Associated with the Complexity of Culturable Fungal and Bacterial Endophyte Communities

Isolated boxwood endophytes have been demonstrated to effectively protect boxwood plants from infection by Calonectria pseudonaviculata (Cps). However, the roles of endophytes as communities in plant defense are not clear. Here, we demonstrated differential tolerance to Cps of English boxwood (Buxus sempervirens ‘Suffruticosa’), an iconic landscape plant and generally regarded as highly susceptible, and its link to endophyte complexity. Fifteen boxwood twig samples were collected in triplicates from three historic gardens—Colonial Williamsburg, George Washington’s Mount Vernon and River Farm, and Virginia Tech’s research farm in Virginia Beach in the summer and fall of 2019. A portion of individual samples was inoculated with Cps under controlled conditions. Significant differences in disease severity were observed among samples but not between the two seasons. Examining the endophyte cultures of the summer samples revealed that bacterial and fungal abundance was negatively and positively correlated with the disease severity. Nanopore metagenomics analysis on genomic DNA of the tolerant and susceptible group representatives confirmed the associations. Specifically, tolerant English boxwood plants had an endophyte community dominated by Bacilli and Betaproteobacteria, while susceptible ones had a distinct endophyte community dominated by Gammaproteobacteria, Actinobacteria, and diverse fungi. These findings may lead to boxwood health management innovations—devising and utilizing cultural practices to manipulate and increase the abundance and performance of beneficial endophytes for enhanced boxwood resistance to Cps

Differential Tolerance to Calonectria pseudonaviculata of English Boxwood Plants Associated with the Complexity of Culturable Fungal and Bacterial Endophyte Communities

Isolated boxwood endophytes have been demonstrated to effectively protect boxwood plants from infection by Calonectria pseudonaviculata (Cps). However, the roles of endophytes as communities in plant defense are not clear. Here, we demonstrated differential tolerance to Cps of English boxwood (Buxus sempervirens ‘Suffruticosa’), an iconic landscape plant and generally regarded as highly susceptible, and its link to endophyte complexity. Fifteen boxwood twig samples were collected in triplicates from three historic gardens—Colonial Williamsburg, George Washington’s Mount Vernon and River Farm, and Virginia Tech’s research farm in Virginia Beach in the summer and fall of 2019. A portion of individual samples was inoculated with Cps under controlled conditions. Significant differences in disease severity were observed among samples but not between the two seasons. Examining the endophyte cultures of the summer samples revealed that bacterial and fungal abundance was negatively and positively correlated with the disease severity. Nanopore metagenomics analysis on genomic DNA of the tolerant and susceptible group representatives confirmed the associations. Specifically, tolerant English boxwood plants had an endophyte community dominated by Bacilli and Betaproteobacteria, while susceptible ones had a distinct endophyte community dominated by Gammaproteobacteria, Actinobacteria, and diverse fungi. These findings may lead to boxwood health management innovations—devising and utilizing cultural practices to manipulate and increase the abundance and performance of beneficial endophytes for enhanced boxwood resistance to Cps