Phytophthora pseudocryptogea Is Associated with Root and Crown Rot of Nursery-Grown Common Sage in Hungary.

In October 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown 2-year-old common sage (Salvia officinalis L. 'Icterina') in two ornamental nurseries in Somogy and Zala counties in Hungary. The disease occurred at a frequency of 15-20% (100 to 150 symptomatic plants in each nursery). A P. cryptogea-like species was isolated consistently from necrotic root collars of many plants on carrot (CA) PARPB agar. Six isolates from the nursery in Zala county and three isolates from the nursery in Somogy county were deposited in the culture collection of Plant Protection Institute (Budapest, Hungary). All developed slightly petaloid colonies on CA agar. Chlamydospores and gametangia were not present in single and dual culture combinations of isolates. Radial colony growth was the fastest at 25°C (6.8 to 7.4 mm/day) and no growth occurred above 34°C. On mycelial discs floating in nonsterile stream water, persistent, nonpapillate, mostly ovoid to obpyriform sporangia (37.4±3.5 to 47.8±4.6 µm long and 22.3±2.6 to 29.2±3.7 µm wide) and hyphal swellings were produced abundantly. Pathogenicity of one selected isolate from each nursery was tested on 3-month-old seedlings of S. officinalis 'Icterina' in 2010. Isolates were grown for 4 weeks at 20°C on autoclaved millet grains moistened with CA broth. Infested and uninfested grains were mixed with autoclaved soil (30 cm3 grain/liter), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth room (20-25°C, 16/8 h dark/light). Pots were flooded for 24 hours on the 1st day and every 2 weeks. All and only treated plants showed symptoms of wilt associated with basal stem and root necrosis within three weeks. The trial was repeated with the same result. The pathogen could be reisolated only from the treated plants. Identity of isolates from nurseries and inoculated plants was confirmed recently by amplification and sequence analysis of the rDNA internal transcribed spacers (ITS) and gene regions of cytochrome c oxidase subunit I (coxI) and ß-tubulin (tub) according to Jung et al. (2017). BLASTn searches showed 100% identity and only 97.3-99.0% similarity to the corresponding sequences of authenthic P. pseudocryptogea and P. cryptogea strains, respectively (e.g., GenBank accession nos. KP288336-KP288342, KP288370-KP288372, KP288386-KP288392, MN872725, MN872776). Sequences of the 9 field isolates were deposited in GenBank under accession nos. OR771701-OR771709 (ITS), OR787508-OR787516 (coxI) and OR787517-OR787525 (tub). P. pseudocryptogea was delineated from P. cryptogea sensu lato (Safaiefarahani et al. 2015), which has been reported from S. officinalis in the United States (Koike 1997), and S. leucantha (Cacciola et al. 2002) and S. officinalis (Garibaldi et al. 2015) in Italy. The known natural hosts of P. pseudocryptogea includes plant species in families other than Lamiaceae (cf. Aloi et al. 2023), but it was pathogenic on the lamiaceous Plectranthus scutellarioides in artificial inoculations (Christova 2020). The pathogen is present in European nurseries (Antonelli et al. 2023). This is the first report of P. pseudocryptogea on S. officinalis in Hungary. The causal agent threatens the production of sages and other ornamentals, and its spread in Hungary should be prevented by proper disease management and phytosanitary actions.

Signals of Systemic Immunity in Plants: Progress and Open Questions.

Systemic acquired resistance (SAR) is a defence mechanism that induces protection against a wide range of pathogens in distant, pathogen-free parts of plants after a primary inoculation. Multiple mobile compounds were identified as putative SAR signals or important factors for influencing movement of SAR signalling elements in Arabidopsis and tobacco. These include compounds with very different chemical structures like lipid transfer protein DIR1 (DEFECTIVE IN INDUCED RESISTANCE1), methyl salicylate (MeSA), dehydroabietinal (DA), azelaic acid (AzA), glycerol-3-phosphate dependent factor (G3P) and the lysine catabolite pipecolic acid (Pip). Genetic studies with different SAR-deficient mutants and silenced lines support the idea that some of these compounds (MeSA, DIR1 and G3P) are activated only when SAR is induced in darkness. In addition, although AzA doubled in phloem exudate of tobacco mosaic virus (TMV) infected tobacco leaves, external AzA treatment could not induce resistance neither to viral nor bacterial pathogens, independent of light conditions. Besides light intensity and timing of light exposition after primary inoculation, spectral distribution of light could also influence the SAR induction capacity. Recent data indicated that TMV and CMV (cucumber mosaic virus) infection in tobacco, like bacteria in Arabidopsis, caused massive accumulation of Pip. Treatment of tobacco leaves with Pip in the light, caused a drastic and significant local and systemic decrease in lesion size of TMV infection. Moreover, two very recent papers, added in proof, demonstrated the role of FMO1 (FLAVIN-DEPENDENT-MONOOXYGENASE1) in conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates after microbial attack and acts as a potent inducer of plant immunity to bacterial and oomycete pathogens in Arabidopsis. These results argue for the pivotal role of Pip and NHP as an important signal compound of SAR response in different plants against different pathogens.

Genetic Diversity and Origins of the Homoploid-Type Hybrid Phytophthora ×alni.

Assessing the process that gives rise to hybrid pathogens is central to understanding the evolution of emerging plant diseases. Phytophthora ×alni, a pathogen of alder, results from the homoploid hybridization of two related species, Phytophthora uniformis and Phytophthora ×multiformis Describing the genetic characteristics of P ×alni should help us understand how reproductive mechanisms and historical processes shaped the population structure of this emerging hybrid pathogen. The population genetic structure of P ×alni and the relationship with its parental species were investigated using 12 microsatellites and one mitochondrial DNA (mtDNA) marker on a European collection of 379 isolates. Populations of P ×alni were dominated by one multilocus genotype (MLG). The frequency of this dominant MLG increased after the disease emergence together with a decline in diversity, suggesting that it was favored by a genetic mechanism such as drift or selection. Combined microsatellite and mtDNA results confirmed that P ×alni originated from multiple hybridization events that involved different genotypes of the progenitors. Our detailed analyses point to a geographic structure that mirrors that observed for P. uniformis in Europe. The study provides more insights on the contribution of P. uniformis, an invasive species in Europe, to the emergence of Phytophthora-induced alder decline. IMPORTANCE: Our study describes an original approach to assess the population genetics of polyploid organisms using microsatellite markers. By studying the parental subgenomes present in the interspecific hybrid P. ×alni, we were able to assess the geographical and temporal structure of European populations of the hybrid, shedding new light on the evolution of an emerging plant pathogen. In turn, the study of the parental subgenomes permitted us to assess some genetic characteristics of the parental species of P. ×alni, P. uniformis, and P ×multiformis, which are seldom sampled in nature. The subgenomes found in P. ×alni represent a picture of the "fossilized" diversity of the parental species.

Virulence Spectra of Hungarian Pyrenophora teres f. teres Isolates Collected from Experimental Fields Show Continuous Variation without Specific Isolate × Barley Differential Interactions.

Pyrenophora teres f. teres (Ptt), the causal agent of net form net blotch (NFNB) disease, is an important and widespread pathogen of barley. This study aimed to quantify and characterize the virulence of Ptt isolates collected from experimental fields of barley in Hungary. Infection responses across 20 barley differentials were obtained from seedling assays of 34 Ptt isolates collected from three Hungarian breeding stations between 2008 and 2018. Twenty-eight Ptt pathotypes were identified. Correspondence analysis followed by hierarchical clustering on the principal components and host-by-pathogen GGE biplots suggested a continuous range of virulence and an absence of specific isolate × barley differential interactions. The isolates were classified into four isolate groups (IG) using agglomerative hierarchical clustering. One IG could be distinguished from other IGs based on avirulence/virulence on one to five barley differentials. Several barley differentials expressed strong resistance against multiple Ptt isolates and may be useful in the development of NFNB-resistant barley cultivars in Hungary. Our results emphasize that the previously developed international barley differential set needs to be improved and adapted to the Hungarian Ptt population. This is the first report on the pathogenic variations of Ptt in Hungary.

Strong genetic differentiation between North American and European populations of Phytophthora alni subsp. uniformis.

Alder decline caused by Phytophthora alni has been one of the most important diseases of natural ecosystems in Europe during the last 20 years. The emergence of P. alni subsp. alni -the pathogen responsible for the epidemic-is linked to an interspecific hybridization event between two parental species: P. alni subsp. multiformis and P. alni subsp. uniformis. One of the parental species, P. alni subsp. uniformis, has been isolated in several European countries and, recently, in North America. The objective of this work was to assess the level of genetic diversity, the population genetic structure, and the putative reproduction mode and mating system of P. alni subsp. uniformis. Five new polymorphic microsatellite markers were used to contrast both geographical populations. The study comprised 71 isolates of P. alni subsp. uniformis collected from eight European countries and 10 locations in North America. Our results revealed strong differences between continental populations (Fst = 0.88; Rst = 0.74), with no evidence for gene flow. European isolates showed extremely low genetic diversity compared with the North American collection. Selfing appears to be the predominant mating system in both continental collections. The results suggest that the European P. alni subsp. uniformis population is most likely alien and derives from the introduction of a few individuals, whereas the North American population probably is an indigenous population.

Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells.

The Human Combinatorial Antibody Library (HuCAL) was screened for antibodies specific to human leukocyte antigen-DR (HLA-DR) that induce programmed death of lymphoma/leukemia cells expressing the target antigen. The active Fab fragments were affinity-matured, and engineered to IgG(4) antibodies of sub-nanomolar affinity. The antibodies exhibited potent in vitro tumoricidal activity on several lymphoma and leukemia cell lines and on chronic lymphocytic leukemia patient samples. They were also active in vivo in xenograft models of non-Hodgkin lymphoma. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms, and was selective to activated/tumor-transformed cells. Although the expression of HLA-DR on normal hematopoietic cells is a potential safety concern, the antibodies caused no long-lasting hematological toxicity in primates, in vivo. Such monoclonal antibodies offer the potential for a novel therapeutic approach to lymphoid malignancies.

Unique clinical and pathological features in HLA-DRB1*0401-restricted MBP 111-129-specific humanized TCR transgenic mice.

Amino acid residues 111-129 represent an immunodominant epitope of myelin basic protein (MBP) in humans with human leukocyte antigen (HLA)-DRB1*0401 allele(s). The MBP 111-129-specific T cell clone MS2-3C8 was repeatedly isolated from a patient with multiple sclerosis (MS), suggesting an involvement of MS2-3C8 T cells in the pathogenesis. To address the pathogenic potential of the MS2-3C8 T cell clone, we generated transgenic (Tg) mice expressing its T cell receptor and restriction element, HLA-DRB1*0401, to examine the pathogenic characteristics of MS2-3C8 Tg T cells by adoptive transfer into HLA-DRB1*0401 Tg mice. In addition to the ascending paralysis typical of experimental autoimmune encephalomyelitis, mice displayed dysphagia due to restriction in jaw and tongue movements and abnormal gait. In accordance with the clinical phenotype, infiltrates of MS2-3C8 Tg T cells and inflammatory lesions were predominantly located in the brainstem and the cranial nerve roots in addition to the spinal cord and spinal nerve roots. Together, these data suggest a pathogenic role of MBP-specific T cells in inflammatory demyelination within the brainstem and cranial nerve roots during the progression of MS. This notion may help to explain the clinical and pathological heterogeneity of MS.

Second-generation peptidomimetic inhibitors of antigen presentation effectively treat autoimmune diseases in HLA-DR-transgenic mouse models.

Peptidomimetic compounds that bind to major histocompatibility complex class II molecules and are resistant to cathepsins can competitively inhibit the presentation of processed protein antigens. Therefore, compounds that bind to autoimmune disease-associated class II molecules are expected to compete with autoantigens for presentation and thereby interrupt the disease process. The first generation of such competitors developed for rheumatoid arthritis-associated HLA-DR molecules, although resistant to cathepsins, has remained sensitive to plasma proteases, and was thus unlikely to be effective in vivo. We have therefore produced a second generation of compounds that are resistant to cathepsins and stable in plasma while maintaining binding affinity for HLA-DR molecules associated with rheumatoid arthritis and multiple sclerosis. Selected compounds of this series are shown to inhibit antigen presentation in vivo, as well as effectively treat collagen induced arthritis and experimental autoimmune encephalomyelitis in HLA-DR transgenic mouse models.

A novel, alternative pathway of apoptosis triggered through class II major histocompatibility complex molecules.

Major histocompatibility complex class II (MHC-II) molecules, in addition to their role of presenting antigen to T lymphocytes, can serve as receptors triggering programmed cell death. MHC-II induced cell death affects activated/tumour transformed cells selectively, and it proceeds without the involvement of caspases, the major proteases of classical apoptosis. Caspase-independent programmed cell death can also be triggered, albeit less effectively, via a series of other cell surface molecules. Here, we discuss the major characteristics, physiological significance, and clinical relevance of caspase-independent apoptotic pathways with particular emphasis on the one induced by MHC-II ligation.

Standard and Swedish variant types of the hybrid alder Phytophthora attacking alder in Hungary.

A new Phytophthora disease of common alder (Alnus glutinosa) similar to that previously reported in several countries in Europe has been observed in Hungary. Based on these earlier studies, the alder Phytophthora was considered likely to be a hybrid between P cambivora and a P fragariae-like species: across Europe a range of new alder Phytophthora is spreading that comprise a range of heteroploid hybrids including a 'standard' hybrid type and several other hybrid types termed 'variants'. Phenotypic and molecular features of the pathogen in Hungary were characterised and compared with isolates from elsewhere. The morphologies of five isolates from one region (Hévíz) resembled the common, 'standard' type, whereas the three isolates from another region (Hanság) exhibited traits similar to those of one of the 'variant' types, ie the Swedish 'variant'. Molecular markers of these two groups of Hungarian isolates also represented a good fit to those of the standard type and the Swedish variant, respectively. Isozyme patterns and profiles of restriction fragments of the entire internal transcribed spacer (ITS) region or mitochondrial DNAs and of RAPD-PCR products did not differ within a group, but distinct polymorphisms were exhibited between the two groups of isolates. Southern analysis of random amplified polymorphic DNA (RAPD) revealed the homologous nature of co-migrating bands of P cambivora and the isolates of alder Phytophthora. Furthermore, restriction fragment profiles of the ITS region of ribosomal DNAs and the mtDNAs were consistent with reported biparental origin of alder Phytophthora. The hybrid status of these continuously evolving pathogens raises many issues and challenges concerning efficient control measures.