Deciphering mycobiota and its functional dynamics in root hairs of Rhododendron campanulatum D. Don through Next-gen sequencing.

The Himalayas provide unique opportunities for the extension of shrubs beyond the upper limit of the tree. However, little is known about the limitation of the biotic factors belowground of shrub growth at these cruising altitudes. To fill this gap, the present study deals with the documentation of root-associated microbiota with their predicted functional profiles and interactions in the host Rhododendron campanulatum, a krummholz species. While processing 12 root samples of R. campanulatum from the sites using Omics we could identify 134 root-associated fungal species belonging to 104 genera, 74 families, 39 orders, 17 classes, and 5 phyla. The root-associated microbiota members of Ascomycota were unambiguously dominant followed by Basidiomycota. Using FUNGuild, we reported that symbiotroph and pathotroph as abundant trophic modes. Furthermore, FUNGuild revealed the dominant prevalence of the saptroptroph guild followed by plant pathogens and wood saprotrophs. Alpha diversity was significantly different at the sites. The heatmap dendrogram showed the correlation between various soil nutrients and some fungal species. The study paves the way for a more in-depth exploration of unidentified root fungal symbionts, their interactions and their probable functional roles, which may serve as an important factor for the growth and conservation of these high-altitude ericaceous plants.

A cryptic root isolate belonging to Geoglossales from potted Rhododendron: its molecular phylogeny and ability to colonize an ericoid mycorrhizal host in vitro.

Although the lifestyle of Geoglossales remains largely unknown, recent advancements have established a hypothesis regarding the ericoid mycorrhizal lifestyle of geoglossoid fungi. In this study, we focused on one isolate of Geoglossales sp. obtained from surface-sterilized roots of potted Rhododendron transiens. We aimed to reveal the phylogenetic position and in vitro colonizing ability of this species in the hair roots of ericoid mycorrhizal plants. Based on our multigene phylogenetic tree, this species is a sister of the genus Sarcoleotia which has not been reported from either other studies or field environment. Its ascocarps could not be obtained, and conspecific sequences were not found in the databases and repositories examined. The Geoglossales sp. colonized the vital rhizodermal cells of blueberries in vitro with hyphal coils. There were relatively large morphological variations of coils consistent with extraradical hyphae; however, overall, the colonization morphologically resembled those by Sarcoleotia globosa and representative ericoid mycorrhizal fungi. The taxonomy and ecological significance of the species remain to be resolved; nevertheless, our results suggest that the ericoid mycorrhizal lifestyle may be widespread within Geoglossales.

Morphologic, molecular, and pathogenic characterization of Phytophthora palmivora isolates causing flower rot on azalea.

The objective of this work was to characterize two Phytophthora palmivora isolates causing floral blight and rot in azalea plants and to evaluate the pathogenicity of this oomycete pathogen on several plant species. Azalea plants with symptoms of flower blight and rot were obtained in the municipality of Holambra-SP. After an attempt of isolation, colonies with Phytophthora characteristics grown only on selective V8 medium. Molecular identification of the isolates was done by amplification and sequencing of ITS and COX2 regions. In the phylogenetic analysis, the azalea isolates clustered with reference isolates of P. palmivora. Morphological characteristics were similar to those described for P. palmivora. Isolates were inoculated in healthy azalea plants and caused leaf blight and floral rot. The pathogen was re-isolated from symptomatic plants completing Koch's postulates. In a host range test, the azalea isolates were able to cause lesions on leaves of vinca, snapdragon, basil, and tomato, and affected both leaves and flowers of geranium. Fruit rot was observed on tomato, potato, sweet pepper, scarlet eggplant, zucchini, cucumber, maroon cucumber, onion, apple, papaya, guava, and carrot. This is the first report of the species P. palmivora causing flower blight and rot in azalea plants in Brazil and probably in the world.

A Novel Group of Rhizobium tumorigenes-Like Agrobacteria Associated with Crown Gall Disease of Rhododendron and Blueberry.

Crown gall is an economically important and widespread plant disease caused by tumorigenic bacteria that are commonly affiliated within the genera Agrobacterium, Allorhizobium, and Rhizobium. Although crown gall disease was reported to occur on rhododendron, literature data regarding this disease are limited. In this study, an atypical group of tumorigenic agrobacteria belonging to the genus Rhizobium was identified as a causative agent of crown gall on rhododendron. Genome analysis suggested that tumorigenic bacteria isolated from rhododendron tumors are most closely related to Rhizobium tumorigenes, a new tumorigenic bacterium discovered recently on blackberry in Serbia. However, R. tumorigenes and novel rhododendron strains belong to separate species and form a homogenous clade within the genus Rhizobium, which we named the "tumorigenes" clade. Moreover, tumorigenic bacteria isolated from rhododendron seem to carry a distinct tumor-inducing (Ti) plasmid, compared with those carried by R. tumorigenes strains and Ti plasmids described thus far. To facilitate rapid identification of bacteria belonging to the "tumorigenes" clade, regardless of whether they are pathogenic or not, a conventional PCR method targeting putative chromosomal gene-encoding flagellin protein FlaA was developed in this study. Finally, our results suggested that this novel group of tumorigenic agrobacteria occurs on blueberry but it cannot be excluded that it is distributed more widely.

Quantifying the hidden costs of imperfect detection for early detection surveillance.

The global spread of pathogens poses an increasing threat to health, ecosystems and agriculture worldwide. As early detection of new incursions is key to effective control, new diagnostic tests that can detect pathogen presence shortly after initial infection hold great potential for detection of infection in individual hosts. However, these tests may be too expensive to be implemented at the sampling intensities required for early detection of a new epidemic at the population level. To evaluate the trade-off between earlier and/or more reliable detection and higher deployment costs, we need to consider the impacts of test performance, test cost and pathogen epidemiology. Regarding test performance, the period before new infections can be first detected and the probability of detecting them are of particular importance. We propose a generic framework that can be easily used to evaluate a variety of different detection methods and identify important characteristics of the pathogen and the detection method to consider when planning early detection surveillance. We demonstrate the application of our method using the plant pathogen Phytophthora ramorum in the UK, and find that visual inspec-tion for this pathogen is a more cost-effective strategy for early detection surveillance than an early detection diagnostic test. This article is part of the theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: epidemic forecasting and control'. This theme issue is linked with the earlier issue 'Modelling infectious disease outbreaks in humans, animals and plants: approaches and important themes'.

Film-Forming Polymers and Surfactants Reduce Infection and Sporulation of Phytophthora ramorum on Rhododendron.

Phytophthora ramorum, cause of sudden oak death and ramorum leaf blight, can persist undetected in infested nurseries. Many conventional fungicides are effective in reducing or delaying symptom expression but some may confound visual detection of infected plants. We tested film-forming polymers (FFPs) and surfactants for their ability to reduce infection and sporulation of P. ramorum on rhododendron. FFPs (Anti-Stress, Moisturin, Nature Shield, Nu-Film, and Vapor Gard) and surfactants (Tergitol, Zonix, and an unregistered AGAE product) were screened in detached-leaf assays. Anti-Stress, Nu-Film, Zonix, and a Nu-Film-Zonix mixture were additionally tested for durability, protection against exposure to infested water, and a reduction in sporulation. FFP effectiveness was retained for at least 3 weeks of exposure to overhead irrigation and rain. Relative to controls, foliar treatments protected rhododendron branches exposed to infested water. No treatments prevented symptom development when applied postinfection but leaves treated with Anti-Stress, Zonix, and the Nu-Film-Zonix mixture produced significantly fewer sporangia relative to controls. Application of FFPs and surfactants to quarantined, potentially infected plants offers a management tool for reducing infection and sporulation but not symptom expression, thereby limiting disease spread without interfering with disease detection.

Characteristics of root-cultivable endophytic fungi from Rhododendron ovatum Planch.

Ericoid mycorrhiza can improve the competitiveness of their host plants at the ecosystem level. The ability of ericoid mycorrhizal fungi to thrive under harsh environmental conditions suggests that they are capable of decomposing plant organic matter. This study aims to characterize 2 strains of root-cultivable endophytic fungi, RooDK1 and RooDK6, from Rhododendron ovatum Planch using colony and hyphal morphology, molecular analysis, observations of mycorrhiza, and investigations of adaptation to different sources of organic matter. Nitrogen utilization was also investigated by assessing protease production and growth on different nitrogen sources. Morphological studies indicated that both species are ericoid mycorrhizal fungi; our molecular studies confirmed RooDK1 as Oidiodendron maius and classified RooDK6 as Pezicula ericae. We observed that only RooDK1 can assist in host plant survival by degrading organic matter. This species also secretes protease and has the highest nitrate reductase activity of these 2 endophytes. Thus, RooDK1 has a greater ability to help the host plants thrive in a harsh habitat.

Sporangia Production Over Time by Phytophthora ramorum on Rhododendron 'Cunningham's White' After Placement at Different Relative Humidities.

We examined the impact of relative humidity (RH) on Phytophthora ramorum sporangia production on Rhododendron 'Cunningham's White'. When diseased plants were maintained under continuous moisture in a mist tent, sporangia were collected from some plants for 22 weeks. More than 3,000 sporangia/leaf/week were collected over the first 3 weeks but levels declined to <100 sporangia/leaf/week after 7 weeks. We also examined the impact of drying on P. ramorum sporangia production. Diseased, detached leaves were maintained in humidity chambers (100, 96.2, 84.5, 74.9, and 56.2% RH) for up to 9 weeks and removed weekly to assess sporulation. For comparison, diseased leaves were harvested from plants maintained with dry foliage or subjected to 10 h of simulated dew nightly. All leaves supported sporulation following 5 weeks at 100% RH, 3 weeks at 96.2% RH, and 1 week at 84.5% RH. All leaves collected from plants subjected to nightly dew supported sporulation for 3 weeks; however, only 66.7% of leaves collected from plants with dry foliage supported sporulation after 1 week. Knowledge of the effects of RH levels on P. ramorum sporulation capacity will prove useful in terms of disease management recommendations and for development of predictive models and pest risk assessments.

Thermal Inactivation of Inoculum of Two Phytophthora Species by Intermittent Versus Constant Heat.

Research on solarization efficacy has examined the critical temperature and minimum exposure time to inactivate soilborne pathogens. Most mathematical models focus on survival of inoculum subjected to a constant heat regime rather than an intermittent heat regime that better simulates field conditions. To develop a more accurate predictive model, we conducted controlled lab experiments with rhododendron leaf disks infested with Phytophthora ramorum and P. pini. Focused in vitro experiments with P. ramorum showed significantly longer survival of inoculum exposed to intermittent versus constant heat, indicating that intermittent heat is less damaging. A similar trend was observed in soil. Damage was evaluated by comparing the reduction in subsequent survival time of inoculum subjected to different intensities of sublethal heat treatments. Inoculum exposure to continuous heat reflected an increasing rate of damage accumulation. Multiple sublethal heat events resulted in a constant rate of damage accumulation which allowed us to calculate total damage as the sum of damage from each heat event. A model including a correction for an intermittent heat regime significantly improved the prediction of thermal inactivation under a temperature regime that simulated field conditions.

[Two sample pooling strategies revealed different root-associated fungal diversity of Rhododendron species].

Objective: ooling of multiple samples is widely used in studying general patterns of microbial communities that are heterogeneously structured in space. Pooling strategies and the number of sequence reads generate biases in the description of diversity and community structure of root-associated fungi. Therefore, we developed a molecular toolbox for fast and accurate identification of the root-associated fungal community of Rododendron species. Methods: Multiple root samples of R. lutescens and R. bureavii were collected for DNA extraction. Effects of two different pooling strategies, i.e. pooling samples prior to vs. post PCR, on fungal species composition were studied by comparing results within host species. Results: Species richness and Shannon-Wiener index of fungal communities of clone library constructed by pooling samples after PCR were higher than that of pooling prior to PCR. High frequency fungal species were detected by both pooling strategies, whereas infrequent species detected by the two strategies differed. Notably, the prior to PCR pooling strategy effectively alleviated the unwanted amplification of host plant sequences when fungal specific primer ITS1f and ITS4 were used. Accumulation curves of fungal species suggested that sequencing at least 50 clones can fully reflect species composition of clone library of the two Rhododendron root-associated fungal community. Conclusion: Clone library constructed by post PCR pooling of samples is better in providing accurate views of fungal diversity and community structure of Rhododendron species.

Soil bacteria as sources of virulence signal providers promoting plant infection by Phytophthora pathogens.

Phytophthora species are known as "plant destroyers" capable of initiating single zoospore infection in the presence of a quorum of chemical signals from the same or closely related species of oomycetes. Since the natural oomycete population is too low to reach a quorum necessary to initiate a disease epidemic, creation of the quorum is reliant on alternate sources. Here, we show that a soil bacterial isolate, Bacillus megaterium Sb5, promotes plant infection by Phytophthora species. In the presence of Sb5 exudates, colonization of rhododendron leaf discs by 12 Phytophthora species/isolates was significantly enhanced, single zoospores of P. nicotianae infected annual vinca and P. sojae race 25 successfully attacked a non-host plant, Nicotiana benthamiana as well as resistant soybean cultivars with RPS1a or RPS3a. Sb5 exudates, most notably the fractions larger than 3 kDa, promoted plant infection by improving zoospore swimming, germination and plant attachment. Sb5 exudates also stimulated infection hypha growth and upregulated effector gene expression. These results suggest that environmental bacteria are important sources of virulence signal providers that promote plant infection by Phytophthora species, advancing our understanding of biotic factors in the environmental component of the Phytophthora disease triangle and of communal infection of plant pathogens.

Bridged Epipolythiodiketopiperazines from Penicillium raciborskii, an Endophytic Fungus of Rhododendron tomentosum Harmaja.

Three new epithiodiketopiperazine natural products [outovirin A (1), outovirin B (2), and outovirin C (3)] resembling the antifungal natural product gliovirin have been identified in extracts of Penicillium raciborskii, an endophytic fungus isolated from Rhododendron tomentosum. The compounds are unusual for their class in that they possess sulfide bridges between α- and ß-carbons rather than the typical α-α bridging. To our knowledge, outovirin A represents the first reported naturally produced epimonothiodiketopiperazine, and antifungal outovirin C is the first reported trisulfide gliovirin-like compound. This report describes the identification and structural elucidation of the compounds by LC-MS/MS and NMR.

Ballistosporomyces changbaiensis sp. nov. and Ballistosporomyces bomiensis sp. nov., two novel species isolated from shrub plant leaves.

Four strains, CB 266(T), CB 272, XZ 44D1(T) and XZ 49D2, isolated from shrub plant leaves in China were identified as two novel species of the genus Ballistosporomyces by the sequence analysis of the small subunit of ribosomal RNA (SSU rRNA), the D1/D2 domains of the large subunit of rRNA (LSU rRNA) and internal transcribed spacer (ITS) + 5.8S rRNA region, and physiological comparisons. Ballistosporomyces changbaiensis sp. nov. (type strain CB 266(T) = CGMCC 2.02298(T) = CBS 10124(T), Mycobank number MB 815700) and Ballistosporomyces bomiensis sp. nov. (type strain XZ 44D1(T) = CGMCC 2.02661(T) = CBS 12512(T), Mycobank number MB 815701) are proposed to accommodate these two new species.

Is the root-colonizing endophyte Acremonium strictum an ericoid mycorrhizal fungus?

In previous investigations, we found that Acremonium strictum (strain DSM 100709) developed intracellular structures with similarity to mycelia of ericoid mycorrhizal fungi in the rhizodermal cells of flax plants and in hair roots of Rhododendron plantlets. A. strictum had also been isolated from roots of ericaceous salal plants and was described as an unusual ericoid mycorrhizal fungus (ERMF). As its mycorrhizal traits were doubted, we revised the hypothesis of a mycorrhizal nature of A. strictum. A successful synthesis of mycorrhiza in hair roots of inoculated ericaceous plants was a first step of evidence, followed by fluorescence microscopy with FUN(®)1 cell stain to observe the vitality of the host cells at the early infection stage. In inoculation trials with in vitro-raised mycorrhiza-free Rhododendron plants in axenic liquid culture and in greenhouse substrate culture, A. strictum was never observed in living hair root cells. As compared to the ERMF Oidiodendron maius and Rhizoscyphus ericae that invaded metabolically active host cells and established a symbiotic unit, A. strictum was only found in cells that were dead or in the process of dying and in the apoplast. In conclusion, A. strictum does not behave like a common ERMF-if it is one at all. A comparison of A. strictum isolates from ericaceous and non-ericaceous hosts could reveal further identity details to generalize or specify our findings on the symbiotic nature of A. strictum. At least, the staining method enables to discern between true mycorrhizal and other root endophytes-a tool for further studies.

Label-free detection of Phytophthora ramorum using surface-enhanced Raman spectroscopy.

In this study, we report on a novel approach for the label-free and species-specific detection of the plant pathogen Phytophthora ramorum from real samples using surface enhanced Raman scattering (SERS). In this context, we consider the entire analysis chain including sample preparation, DNA isolation, amplification and hybridization on SERS substrate-immobilized adenine-free capture probes. Thus, the SERS-based detection of target DNA is verified by the strong spectral feature of adenine which indicates the presence of hybridized target DNA. This property was realized by replacing adenine moieties in the species-specific capture probes with 2-aminopurine. In the case of the matching capture and target sequence, the characteristic adenine peak serves as an indicator for specific DNA hybridization. Altogether, this is the first assay demonstrating the detection of a plant pathogen from an infected plant material by label-free SERS employing DNA hybridization on planar SERS substrates consisting of silver nanoparticles.

A revision of the history of the Colletotrichum acutatum species complex in the Nordic countries based on herbarium specimens.

Herbaria collections containing plants with disease symptoms are highly valuable, and they are often the only way to investigate outbreaks and epidemics from the past as the number of viable isolates in culture collections is often limited. Species belonging to the Colletotrichum acutatum complex infect a range of important crops. As members of the C. acutatum complex are easily confused with other Colletotrichum species, molecular methods are central for the correct identification. We performed molecular analyses on 21 herbaria specimens, displaying anthracnose symptoms, collected in Norway and Denmark before the first confirmed findings of C. acutatum complex members in this region. Sequencing parts of the fungal ITS regions showed that members of the species complex were present in 13 of the 21 specimens collected in different parts of Norway and Denmark between 1948 and 1991, representing seven plant hosts (three cherry species, apple, raspberry and rhododendron). This is the first time herbarium specimens have been used to study these pathogens under Nordic conditions. Differences in the ITS sequences suggest the presence of different genotypes within the complex, indicating a well-established population.

EFFICACY OF FUNGICIDES AGAINST CALONECTRIA PAUCIRAMOSA IN POT AZALEA.

Calonectria (formerly Cylindrocladium) infection of pot azalea (Rhododendron simsii Planch) is an important disease problem in which usually one or two of the four plants per pot show progressing leaf and especially stem lesions, leading to mortality of the respective plant and rendering the pot unmarketable. This may occur in a later stage of the growing season, leading to significant commercial losses. The main objective of this study was to test a range of fungicides for their efficacy against this pathogen. To test the fungicides, a bioassay was first developed in which mycelium and conidiospores of the pathogen were produced on Potato Dextrose Agar, blended in water, and dilutions of the resulting suspension inoculated at the base of 11-week-old cuttings three weeks after they had been trimmed. Disease progression was monitored up to 7 weeks post inoculation and a disease index on a scale of 0 to 3 was established. In the actual efficacy trial, the following fungicides (with corresponding active ingredient(s)) were tested as preventive treatments: Topsin M 70 WG (thiophanate-methyl), Sporgon (prochloraz), Signum (boscalid+pyraclostrobin), Switch (cyprodinyl+fludioxonil), Flint 50WG (trifloxystrobin), Ortiva Top (azoxystrobin+difenoconazole) and Fungaflor (imazalil). Disease expression started after about 2 weeks, increased approximately 1 index level, and leveled off 5 weeks after inoculation. The best control was observed with Sporgon, Ortiva Top and Signum. Switch produced intermediate effects and insufficient control was observed with Topsin, Flint and Fungaflor. These results explain why specific standard fungicide treatments, such as those with Topsin, fail to control the disease, while they can be effective against a different Calonectria species such as C. pseudonaviculata, the cause of boxwood blight.

Different distribution patterns between putative ercoid mycorrhizal and other fungal assemblages in roots of Rhododendron decorum in the Southwest of China.

Fungal diversity within plant roots is affected by several factors such as dispersal limitation, habitat filtering, and plant host preference. Given the differences in life style between symbiotic and non-symbiotic fungi, the main factors affecting these two groups of fungi may be different. We assessed the diversity of root associated fungi of Rhododendron decorum using internal transcribed spacer (ITS) sequencing and terminal restriction fragment length polymorphism (T-RFLP) analysis, and our aim was to evaluate the role of different factors in structuring ericoid mycorrhizal (ERM) and non-ericoid mycorrhizal (NEM) fungal communities. Thirty-five fungal operational taxonomic units (OTUs) were found in roots of R. decorum, of which 25 were putative ERM fungal species. Of the two main groups of known ERM, helotialean fungi were more abundant and common than sebacinalean species. Geographic and host patterning of the fungal assemblages were different for ERM and NEM. The distribution of putative ERM fungal terminal restriction fragments (TRFs) showed that there were more common species within ERM than in the NEM fungal assemblages. Results of Mantel tests indicated that the composition of NEM fungal assemblages correlated with geographic parameters while ERM fungal assemblages lacked a significant geographic pattern and instead were correlated with host genotype. Redundancy analysis (RDA) showed that the NEM fungal assemblages were significantly correlated with latitude, longitude, elevation, mean annual precipitation (MAP), and axis 2 of a host-genetic principle component analysis (PCA), while ERM fungal assemblages correlated only with latitude and axis 1 of the host-genetic PCA. We conclude that ERM and NEM assemblages are affected by different factors, with the host genetic composition more important for ERM and geographic factors more important for NEM assemblages. Our results contribute to understanding the roles of dispersal limitation, abiotic factors and biotic interactions in structuring fungal communities in plant roots.

Phytophthora aquimorbida sp. nov. and Phytophthora taxon 'aquatilis' recovered from irrigation reservoirs and a stream in Virginia, USA.

Two distinct subgroups (L2 and A(-2)) were recovered from irrigation reservoirs and a stream in Virginia, USA. After molecular, morphological and physiological examinations, the L2 subgroup was named Phytophthora aquimorbida and the A(-2) designated as Phytophthora taxon 'aquatilis'. Both taxa are homothallic. P. aquimorbida is characterized by its noncaducous and nonpapillate sporangia, catenulate and radiating hyphal swellings and thick-walled plerotic oospores formed in globose oogonia mostly in the absence of an antheridium. P. taxon 'aquatilis' produces plerotic oospores in globose oogonia mostly with a paragynous antheridium. It has semi-papillate, caducous sporangia with variable pedicels, but it does not have hyphal swelling. Analyses of ITS, CO1, ß-tubulin and NADH1 sequences revealed that P. aquimorbida is closely related to P. hydropathica, P. irrigata and P. parsiana, and P. taxon 'aquatilis' is related to P. multivesiculata. The optimum temperature for culture growth is 30 and 20 C for P. aquimorbida and P. taxon 'aquatilis' respectively. Both taxa were pathogenic to rhododendron plants and caused root discoloration, pale leaves, wilting, tip necrosis and dieback. Their plant biosecurity risk also is discussed.

Compost suppressiveness against Phytophthora spp. on Skimmia japonica and azalea.

Suppression of soil-borne plant diseases with composts has been widely studied. Composts have been found to be suppressive against several soil-borne pathogens in various cropping systems. Ornamental plants are generally cultivated in pots, allowing the use of suppressive substrates to control zoospore-producing pathogens, like Phytophthora sp. The objective of the present work was to assess compost suppressiveness against Phytophthora cinnamomi on Rhododendron spp., and against Phytophthora nicotianae, an emerging pathogen on Skimmia japonica. A municipal compost that showed a good suppressive activity in previous trials on vegetable crops was used. Compost was mixed at 10, 20 e 40% (v/v) with a commercial peat substrate, used as control. Substrates have been inoculated at 1g/l dosage of wheat and hemp kernels of Phytophthora spp. and after one week 15-20 plants were transplanted for each treatment in 2 liters volume pots and placed in greenhouse at 20 degrees C. A chemical control (Metalaxil-M) was also used. Diseased plants were assessed weekly after transplanting and above-ground biomass of plants was assessed at the end of the trials. Results showed a significant disease control when compost was used at 20-40% on S. japonica, without showing any phytotoxic effect. Disease suppression was shown at 40% on azalea, but compost was slightly phytotoxic on plants. The use of compost based substrates can be a suitable strategy for controlling soil-borne diseases on ornamentals, but results depend also on alkalinity tolerance of plants.