Molecular-Phylogenetic Characterization of Xanthomonas hortorum pv. pelargonii Strains Causing Leaf Spot of Geraniums in Iran.

Bacterial blight and leaf spot of geraniums is a destructive disease of cultivated Pelargonium species around the world. During 2020 to 2021, surveys were conducted in seven geranium-growing provinces of Iran to monitor the status of bacterial blight and leaf spot disease. The disease was observed in six surveyed provinces varying in the extent of occurrence and severity. Twenty-two gram-negative pale-yellow bacterial strains resembling members of Xanthomonas were isolated from symptomatic leaves and stems. Pathogenicity and host range assays showed that the bacterial strains were pathogenic on Pelargonium grandiflorum, P. graveolens, P. peltatum, and P. zonale. All strains were positive for a PCR test using the primer pair XcpM1/XcpM2, which is specific for X. hortorum pv. pelargonii. Phylogenetic analysis using the sequences of gyrB and lepA genes showed that the 22 strains clustered in a clade among the sequences of X. hortorum pv. pelargonii strains retrieved from GenBank but were distinct from the other pathovars of X. hortorum. BOX-PCR-based fingerprinting using BOX-A1R primer revealed that the strains isolated in this study were grouped into two clusters, while no distinct correlation was observed between the host/area of isolation and BOX-PCR fingerprinting. None of the strains obtained in this study nor the reference strain of the pathogen produced bacteriocin against each other. Results obtained in this study shed light on the geographic distribution, taxonomic status, and host range of the bacterial blight and leaf spot pathogen of geraniums in Iran, paving the path for further research on disease management.

Molecular Profiles of Contrasting Shade Response Strategies in Wild Plants: Differential Control of Immunity and Shoot Elongation.

Plants growing at high densities elongate their shoots to reach for light, a response known as the shade avoidance syndrome (SAS). Phytochrome-mediated detection of far-red light reflection from neighboring plants activates growth-promoting molecular pathways leading to SAS However, it is unknown how plants that complete their life cycle in the forest understory and are shade tolerant prevent SAS when exposed to shade. Here, we show how two wild Geranium species from different native light environments regulate contrasting responses to light quality cues. A comparative RNA sequencing approach unveiled the molecular underpinnings of their contrasting growth responses to far-red light enrichment. It also identified differential phytochrome control of plant immunity genes and confirmed that far-red enrichment indeed contrastingly affects resistance against Botrytis cinerea between the two species. Furthermore, we identify a number of candidate regulators of differential shade avoidance. Three of these, the receptor-like kinases FERONIA and THESEUS1 and the non-DNA binding bHLH protein KIDARI, are functionally validated in Arabidopsis thaliana through gene knockout and/or overexpression studies. We propose that these components may be associated with either showing or not showing shade avoidance responses.

Streptomyces geranii sp. nov., a novel endophytic actinobacterium isolated from root of Geranium carolinianum L.

A novel endophytic actinomycete, designated A301T, was isolated from the root of Geranium carolinianum Linn collected from Mount Emei in China and characterized using a polyphasic approach. Growth occurred at 10-37 °C, pH 6-11 and in the presence of 0-5 % NaCl (w/v). Strain A301T contained ll-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The whole-cell hydrolysates included galactose and ribose. The predominant menaquinones were MK-9(H6) and MK-9(H8). The major cellular fatty acids were C15 : 0, C16 :  0, anteiso-C15 : 0 and iso-C16 : 0. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, two unidentified phospholipids, three unidentified lipids and two unidentified aminophospholipids. Strain A301T shared the highest 16S rRNA gene sequence similarity to Streptomyces cinereorubersubsp. fructofermentans NBRC 15396T (98.1 %) and Streptomyces turgidiscabies ATCC 700248T (98.1 %). The DNA-DNA relatedness values between strain A301T and the two above-mentioned members of the genus Streptomyces were 42.6 % and 47.2 %, respectively. The G+C content of the DNA was 70.5 mol%. On the basis of the polyphasic approach and DNA-DNA hybridization data, strain A301T represents a novel species within the genus Streptomyces, for which the name Streptomyces geranii sp. nov. is proposed. The type strain is A301T (=CGMCC 4.7422T=JCM 32177T).

Effects of arbuscular mycorrhizal fungi and maternal plant sex on seed germination and early plant establishment.

UNLABELLED: • PREMISE OF THE STUDY: Arbuscular mycorrhizal fungi usually enhance overall plant performance, yet their effects on seed germination and early plant establishment, crucial steps in plant cycles, are generally overlooked. In gynodioecious species, sexual dimorphism in these traits has been reported, with females producing seeds that germinate at a faster rate than seeds from hermaphrodites.• METHODS: Using the gynodioecious plant Geranium sylvaticum, I investigated in a greenhouse experiment whether the presence of arbuscular mycorrhizal spores affects seed germination and early plant establishment, examining at the same time whether the sex of the mother producing the seeds also influences these parameters and whether sex-specific interactions between these two factors exist.• KEY RESULTS: The presence of arbuscular mycorrhizal spores in the soil decreased seed germination, did not affect plant survival, but did increase plant growth. Moreover, no significant differences in seed traits were detected between the sexes of the plants producing the seeds.• CONCLUSIONS: This study demonstrates that arbuscular mycorrhizal fungi may have contrasting effects for plants during early life stages and that mycorrhizal effects can take place even at the precolonization stage.

Transgenerational effects of plant sex and arbuscular mycorrhizal symbiosis.

In gynodioecious plants, females are predicted to produce more and/or better offspring than hermaphrodites in order to be maintained in the same population. In the field, the roots of both sexes are usually colonized by arbuscular mycorrhizal (AM) fungi. Transgenerational effects of mycorrhizal symbiosis are largely unknown, although theoretically expected. We examined the maternal and paternal effects of AM fungal symbiosis and host sex on seed production and posterior seedling performance in Geranium sylvaticum, a gynodioecious plant. We hand-pollinated cloned females and hermaphrodites in symbiosis with AM fungi or in nonmycorrhizal conditions and measured seed number and mass, and seedling survival and growth in a glasshouse experiment. Females produced more seeds than hermaphrodites, but the seeds did not germinate, survive or grow better. Mycorrhizal plants were larger, but did not produce more seeds than nonmycorrhizal plants. Transgenerational parental effects of AM fungi were verified in seedling performance. This is the first study to show transgenerational mycorrhiza-mediated parental effects in a gynodioecious species. Mycorrhizal symbiosis affects plant fitness mainly through female functions with enduring effects on the next generation.

Effect of light on in vivo urediniospore germination, lesion development and sporulation of Puccinia hemerocallidis on daylily and Puccinia pelargoniizonalis on geranium.

The presence of rusts of daylily and geranium caused respectively by Puccinia hemerocallidis and P. pelargoniizonalis can result in reduced value of these ornamental crops. Experiments were conducted to determine the effects of fluorescent light and sunlight on urediniospore germination, germ tube elongation, lesion development and sporulation of the two fungal pathogens on detached leaves and whole plants. Exposure of dry or hydrated urediniospores of P. hemerocallidis to cool white fluorescent light (600 µmol s(-1) m(-2)) or to sunlight (950-1910 µmol s(-1) m(-2)) for 2 h or 4 h significantly reduced germination and germ tube elongation on detached daylily leaves. Germination but not germ tube elongation of hydrated urediniospores of P. pelargoniizonalis on detached geranium leaves was significantly reduced when exposed to fluorescent light for 2 h or 4 h. A 4 h exposure to either light source significantly reduced lesion development of P. hemerocallidis on detached daylily leaves with fewer lesions developing from hydrated compared to dry urediniospores. Sunlight exposures of 1 h and 2 h of hydrated and dry urediniospores respectively significantly reduced lesion development by either fungus on whole plants. Increasing exposure to fluorescent light negatively affected sporulation of P. hemerocallidis and P. pelargoniizonalis. Complete suppression of sporulation was not observed for either fungus with up to a 24 h exposure to fluorescent light. Light exposure negatively affected disease development by P. hemerocallidis and P. pelargoniizonalis. Exposure to high light intensities may affect spread of rust diseases on ornamental plants.

Moderate temperature fluctuations rapidly reduce the viability of Ralstonia solanacearum race 3, biovar 2, in infected geranium, tomato, and potato plants.

Most Ralstonia solanacearum strains are tropical plant pathogens, but race 3, biovar 2 (R3bv2), strains can cause bacterial wilt in temperate zones or tropical highlands where other strains cannot. R3bv2 is a quarantine pathogen in North America and Europe because of its potential to damage the potato industry in cooler climates. However, R3bv2 will not become established if it cannot survive temperate winters. Previous experiments showed that in water at 4°C, R3bv2 does not survive as long as native U.S. strains, but R3bv2 remains viable longer than U.S. strains in potato tubers at 4°C. To further investigate the effects of temperature on this high-concern pathogen, we assessed the ability of R3bv2 and a native U.S. strain to survive typical temperate winter temperature cycles of 2 days at 5°C followed by 2 days at -10°C. We measured pathogen survival in infected tomato and geranium plants, in infected potato tubers, and in sterile water. The population sizes of both strains declined rapidly under these conditions in all three plant hosts and in sterile water, and no culturable R. solanacearum cells were detected after five to seven temperature cycles in plant tissue. The fluctuations played a critical role in loss of bacterial viability, since at a constant temperature of -20°C, both strains could survive in infected geranium tissue for at least 6 months. These results suggest that even when sheltered in infected plant tissue, R3bv2 is unlikely to survive the temperature fluctuations typical of a northern temperate winter.

Mycorrhizal benefit differs among the sexes in a gynodioecious species.

Both plant sex and arbuscular mycorrhizal (AM) symbiosis influence resource acquisition and allocation in plants, but the interaction between these two components is not well established. As the different plant sexes differ in their resource needs and allocation patterns, it is logical to presume that they might differ in their relationship with AM as well. We investigate whether the association with AM symbiosis is different according to the host plant sex in the gynodioecious Geranium sylvaticum, of which, besides female and hermaphrodite plants, intermediate plants are also recognized. Specifically, we examine the effects of two different AM fungi in plant mass allocation and phosphorus acquisition using a factorial greenhouse/common garden experiment. Cloned G. sylvaticum material was grown in symbiosis with AM fungi or in non-mycorrhizal condition. We evaluated both the symbiotic plant benefit in terms of plant mass and plant P content and the fungal benefit in terms of AM colonization intensity in the plant roots and spore production. Our results suggest that G. sylvaticum plants benefit from the symbiosis with both AM fungal species tested but that the benefits gained from the symbiosis depend on the sex of the plant and on the trait investigated. Hermaphrodites suffered most from the lack of AM symbiosis as the proportion of flowering plants was dramatically reduced by the absence of AM fungi. However, females and intermediates benefited from the symbiosis relatively more than hermaphrodites in terms of higher P acquisition. The two AM fungal species differed in the amount of resources accumulated, and the fungal benefit was also dependent on the sex of the host plant. This study provides the first evidence of sex-specific benefits from mycorrhizal symbiosis in a gynodioecious plant species.

Transstadial transmission of Pythium in Bradysia impatiens and lack of adult vectoring capacity.

Fungus gnats have been shown to transmit a variety of plant-pathogenic fungi that produce aerial dispersal stages. However, few studies have examined potential interactions between fungus gnats and oomycetes, including Pythium spp. A series of laboratory experiments were conducted to determine whether fungus gnat adults are vectors of several common greenhouse Pythium spp., including Pythium aphanidermatum, P. irregulare, and P. ultimum. An additional objective was to determine whether P. aphanidermatum can be maintained transstadially in the gut of a fungus gnat larva through the pupal stadium to be transmitted by the subsequent adult. Adult fungus gnats did not pick up infectious Pythium propagules from diseased plants and transmit them to healthy plants in any experiment. Species-specific primers and a probe for real-time polymerase chain reaction were developed to detect the presence of P. aphanidermatum DNA in fungus gnat tissue samples. P. aphanidermatum DNA was detectable in the larval and pupal stages; however, none was detected in adult fungus gnats. These results are in agreement with previous studies that have suggested that adult fungus gnats are unlikely vectors of Pythium spp.

Arbuscular mycorrhizal fungi change host plant DNA methylation systemically.

DNA methylation is an important epigenetic mechanism regulating gene expression in plants. DNA methylation has been shown to vary among species and also among plant tissues. However, no study has evaluated whether arbuscular mycorrhizal (AM) fungi affect DNA methylation levels in a tissue-specific manner. We investigated whether symbiosis with AM fungi affects DNA methylation in the host, focusing on different plant tissues (roots versus leaves) and across time. We carried out a 6-month pot experiment using Geranium robertianum in symbiosis with the AM fungus Funneliformis mosseae. Our results show that the pattern of total DNA methylation differed between leaves and roots and was related to when plants were harvested, confirming that DNA methylation is a process that occurs dynamically throughout an organism's lifetime. More importantly, the presence of AM fungus in roots of our experimental plants had a positive effect on total DNA methylation in both tissues. This study shows that colonisation by AM fungi can affect DNA methylation levels in their hosts and that plant DNA methylation varies in an age- and tissue-specific manner.

Root traits and belowground herbivores relate to plant-soil feedback variation among congeners.

Plant-soil feedbacks contribute to vegetation dynamics by species-specific interactions between plants and soil biota. Variation in plant-soil feedbacks can be predicted by root traits, successional position, and plant nativeness. However, it is unknown whether closely related plant species develop more similar plant-soil feedbacks than more distantly related species. Where previous comparisons included plant species from distant phylogenetic positions, we studied plant-soil feedbacks of congeneric species. Using eight intra-continentally range-expanding and native Geranium species, we tested relations between phylogenetic distances, chemical and structural root traits, root microbiomes, and plant-soil feedbacks. We show that root chemistry and specific root length better predict bacterial and fungal community composition than phylogenetic distance. Negative plant-soil feedback strength correlates with root-feeding nematode numbers, whereas microbiome dissimilarity, nativeness, or phylogeny does not predict plant-soil feedbacks. We conclude that root microbiome variation among congeners is best explained by root traits, and that root-feeding nematode abundances predict plant-soil feedbacks.

Molecular diagnostic procedures for production of pathogen-free propagation material.

Production of disease-free propagation material is a major means of controlling most bacterial diseases of plants, particularly when neither resistant clones nor effective chemical treatments are available. For this purpose sensitive, specific and rapid detection methods are required. The advent of molecular biology and, in particular, the polymerase chain reaction (PCR) has opened new ways for the characterization and identification of plant pathogens and the development of disease-management strategies. PCR-based detection methods rely on the development of primers for the specific detection of the pathogen. The use of pathogenicity genes as targets for primer design is the preferred procedure for obtaining specific primers but other procedures may also be useful for this purpose. In the present review we describe four examples of procedures for detecting four important bacterial pathogens in Israel: Erwinia herbicola pv gypsophilae in gypsophila, Xanthomonas campestris pv pelargonii in geranium, Agrobacterium tumefaciens in asters and roses, and Xanthomonas campestris pv campestris in crucifers. Procedures for constructing specific PCR primers for each bacterium are illustrated and discussed as well as the combination of PCR with other methods.

Variable mycorrhizal benefits on the reproductive output of Geranium sylvaticum, with special emphasis on the intermediate phenotype.

In several gynodioecious species, intermediate sex between female and hermaphrodite has been reported, but few studies have investigated fitness parameters of this intermediate phenotype. Here, we examined the interactions between plant sex and arbuscular mycorrhizal (AM) fungal species affecting the reproductive output of Geranium sylvaticum, a sexually polymorphic plant species with frequent intermediate sexes between females and hermaphrodites, using a common garden experiment. Flowering phenology, AM colonisation levels and several plant vegetative and reproductive parameters, including seed and pollen production, were measured. Differences among sexes were detected in flowering, fruit set, pollen production and floral size. The two AM species used in the present work had different effects on plant fitness parameters. One AM species increased female fitness through increasing seed number and seed mass, while the other species reduced seed mass in all sexes investigated. AM fungi did not affect intermediate and hermaphrodite pollen content in anthers. The three sexes in G. sylvaticum did not differ in their reproductive output in terms of total seed production, but hermaphrodites had potentially larger fathering ability than intermediates due to higher anther number. The ultimate female function--seed production--did not differ among the sexes, but one of the AM fungi used potentially decreased host plant fitness. In addition, in the intermediate sex, mycorrhizal symbiosis functioned similarly in females as in hermaphrodites.

Subsurface examination of a foliar biofilm using scanning electron- and focused-ion-beam microscopy.

The dual beam scanning electron microscope, equipped with both a focused ion- and scanning electron-beam (FIB SEM) is a novel tool for the exploration of the subsurface structure of biological tissues. The FIB can remove a predetermined amount of material from a selected site to allow for subsurface exploration and when coupled with SEM or scanning ion-beam microscopy (SIM) could be suitable to examine the subsurface structure of bacterial biofilms on the leaf surface. The suitability of chemical and cryofixation was examined for use with the FIB SEM to examine bacterial biofilms on leaf surfaces. The biological control agent, Burkholderia pyroccinia FP62, that rapidly colonizes the leaf surface and forms biofilms, was inoculated onto geranium leaves and incubated in a greenhouse for 7 or 14 days. Cryofixation was not suitable for examination of leaf biofilms because it created a frozen layer over the leaf surface that cracked when exposed to the electron beam and the protective cap required for FIB milling could not be accurately deposited. With chemically fixed samples, it was possible to precisely FIB mill a single cross section (5µm) or sequential cross sections from a single site without any damage to the surrounding surface. Biofilms, 7 days post-inoculation (DPI), were composed of 2-5 bacterial cell layers while biofilms 14 DPI ranged from 5 to greater than 30 cell layers. Empty spaces between bacteria cells in the subsurface structure were observed in biofilms 7- and 14-DPI. Sequential cross sections inferred that the empty spaces were often continuous between FP62 cells and could possibly make up a network of channels throughout the biofilm. FIB SEM was a useful tool to observe the subsurface composition of a foliar biofilm.