Using Verticillium albo-atrum WCS850 to control Dutch elm disease

Approximately 100 years after the first introduction of Dutch elm disease (DED) in Europe, an effective commercial biocontrol product is available to protect susceptible elm trees against DED transmission by beetles. Injection of trees with conidiospores of the fungus Verticilium albo-atrum isolate WCS850, product name DutchTrig®, reduces infection to less than 0.2% of treated trees. This biocontrol agent, its mode of action, application, efficacy and limitations in controlling DED are described in this chapter.

Priming of Plant Defenses against Ophiostoma novo-ulmi by Elm (Ulmus minor Mill.) Fungal Endophytes

Some fungal endophytes of forest trees are recognized as beneficial symbionts against stresses. In previous works, two elm endophytes from the classes Cystobasidiomycetes and Eurotiomycetes promoted host resistance to abiotic stress, and another elm endophyte from Dothideomycetes enhanced host resistance to Dutch elm disease (DED). Here, we hypothesize that the combined effect of these endophytes activate the plant immune and/or antioxidant system, leading to a defense priming and/or increased oxidative protection when exposed to the DED pathogen Ophiostoma novo-ulmi. To test this hypothesis, the short-term defense gene activation and antioxidant response were evaluated in DED-susceptible (MDV1) and DED-resistant (VAD2 and MDV2.3) Ulmus minor genotypes inoculated with O. novo-ulmi, as well as two weeks earlier with a mixture of the above-mentioned endophytes. Endophyte inoculation induced a generalized transient defense activation mediated primarily by salicylic acid (SA). Subsequent pathogen inoculation resulted in a primed defense response of variable intensity among genotypes. Genotypes MDV1 and VAD2 displayed a defense priming driven by SA, jasmonic acid (JA), and ethylene (ET), causing a reduced pathogen spread in MDV1. Meanwhile, the genotype MDV2.3 showed lower defense priming but a stronger and earlier antioxidant response. The defense priming stimulated by elm fungal endophytes broadens our current knowledge of the ecological functions of endophytic fungi in forest trees and opens new prospects for their use in the biocontrol of plant diseases.

Cryopreservation and Micropropagation Methods for Conservation of Genetic Resources of Ulmus laevis and Ulmus glabra

Elms are threatened by Dutch elm disease, and conservation methods are needed to protect their genetic diversity. Cryopreservation of dormant buds allows large numbers of genotypes to be conserved with small space requirements and minimal upkeep. Cryopreservation through slow controlled cooling was tested for both elm species native to Finland, Ulmus glabra and Ulmus laevis. Regeneration of the thawed buds by micropropagation was studied on different basal media and using different growth regulators. Multiple surface sterilisation methods were tried out for bud explants. The multiplication of U. glabra was investigated with Driver and Kuniyuki walnut medium with either 0.5 mg/L meta-topolin or 0.5 mg/L 6-benzylaminopurine. Rooting with short indole-6-butyric acid induction in liquid medium and direct transplantation of the shoots to peat ex vitro after induction were tested. For initiation, either Murashige and Skoog or Driver and Kuniyuki walnut medium with 0.02 mg/L gibberellic acid 4 + 7 and 0.5 mg/L 6-benzylaminopurine were found to best promote shoot formation. Surface sterilisation remains the most challenging step. No significant differences were found between the multiplication media in either shoot production or rooting success. Rooting by direct transplanting was achieved in both species, but further development is required before application on a larger scale. With further improvements to sterilisation success especially in U. glabra, the method can be applied to the conservation of genetic resources of both U. laevis and U. glabra, and knowledge of regeneration success can be used to design the cryoconservation plan and optimise the sampling.

Complexities underlying the breeding and deployment of Dutch elm disease resistant elms

Dutch elm disease (DED) is a vascular wilt disease caused by the pathogens Ophiostoma ulmi and Ophiostoma novo-ulmi with multiple ecological phases including pathogenic (xylem), saprotrophic (bark) and vector (beetle flight and beetle feeding wound) phases. Due to the two DED pandemics during the twentieth century the use of elms in landscape and forest restoration has declined significantly. However new initiatives for elm breeding and restoration are now underway in Europe and North America. Here we discuss complexities in the DED ‘system’ that can lead to unintended consequences during elm breeding and some of the wider options for obtaining durability or ‘field resistance’ in released material, including (1) the phenotypic plasticity of disease levels in resistant cultivars infected by O. novo-ulmi; (2) shortcomings in test methods when selecting for resistance; (3) the implications of rapid evolutionary changes in current O. novo-ulmi populations for the choice of pathogen inoculum when screening; (4) the possibility of using active resistance to the pathogen in the beetle feeding wound, and low attractiveness of elm cultivars to feeding beetles, in addition to resistance in the xylem; (5) the risk that genes from susceptible and exotic elms be introgressed into resistant cultivars; (6) risks posed by unintentional changes in the host microbiome; and (7) the biosecurity risks posed by resistant elm deployment. In addition, attention needs to be paid to the disease pressures within which resistant elms will be released. In the future, biotechnology may further enhance our understanding of the various resistance processes in elms and our potential to deploy trees with highly durable resistance in elm restoration. Hopefully the different elm resistance processes will prove to be largely under durable, additive, multigenic control. Elm breeding programmes cannot afford to get into the host–pathogen arms races that characterise some agricultural host–pathogen systems.

An Efficient Strategy for Obtaining Mutants by Targeted Gene Deletion in Ophiostoma novo-ulmi

The dimorphic fungus Ophiostoma novo-ulmi is the highly aggressive pathogen responsible for the current, highly destructive, pandemic of Dutch elm disease (DED). Genome and transcriptome analyses of this pathogen previously revealed that a large set of genes expressed during dimorphic transition were also potentially related to plant infection processes, which seem to be regulated by molecular mechanisms different from those described in other dimorphic pathogens. Then, O. novo-ulmi can be used as a representative species to study the lifestyle of dimorphic pathogenic fungi that are not shared by the “model species” Candida albicans and Ustilago maydis. In order to gain better knowledge of molecular aspects underlying infection process and symptom induction by dimorphic fungi that cause vascular wilt disease, we developed a high-throughput gene deletion protocol for O. novo-ulmi. The protocol is based on transforming a Δmus52 O. novo-ulmi mutant impaired for non-homologous end joining (NHEJ) as the recipient strain, and transforming this strain with the latest version of OSCAR plasmids. The latter are used for generating deletion constructs containing the toxin-coding Herpes simplex virus thymidine kinase (HSVtk) gene which prevents ectopic integration of the T-DNA in Ophiostoma DNA. The frequency of gene deletion by homologous recombination (HR) at the ade1 locus associated with purine nucleotide biosynthesis was up to 77.8% in the Δmus52 mutant compared to 2% in the wild-type (WT). To validate the high efficiency of our deletion gene methodology we deleted ade7, which also belongs to the purine nucleotide pathway, as well as bct2, ogf1, and opf2 which encode fungal binuclear transcription factors (TFs). The frequency of gene replacement by HR for these genes reached up to 94%. We expect that our methodology combining the use of NHEJ deficient strains and OSCAR plasmids will function with similar high efficiencies for other O. novo-ulmi genes and other filamentous fungi.

Chlorostroma vestlandicum (Xylariales): first records of the rare ascomycete in Russia

In this paper, for the first time in Russia we report on four new occurrences of the rare xylarialean ascomycete Chlorostroma vestlandicumrevealed in the course of mycological surveys of the Northwestern European Russia. Description and colour illustrations of the material studied are provided. Dutch Elm Disease as the major possible threat for the species is discussed. The species is recommended for inclusion in the next editions of the regional Red Data Books of Leningrad, Novgorod, and Pskov Regions.

Enhanced Outcrossing, Directional Selection and Transgressive Segregation Drive Evolution of Novel Phenotypes in Hybrid Swarms of the Dutch Elm Disease Pathogen Ophiostoma novo-ulmi

In the 1970s, clones of the two subspecies of Ophiostoma novo-ulmi, subsp. americana (SSAM) and subsp. novo-ulmi (SSNU) began to overlap in Europe, resulting in hybrid swarms. By 1983–1986, hybrids with high, SSAM-like growth and pathogenic fitness comprised ~75% of popula-tions at Limburg, Netherlands and Orvieto, Italy. We resampled these populations in 2008 to examine trends in hybrid fitness traits. Since preliminary sampling in 1979–1980, MAT-1 locus frequency had increased from ~0% to ~32% at Orvieto and 5% to ~43% at Limburg, and vegeta-tive incompatibility type frequency had changed from near clonal to extremely diverse at both sites. This represents an enormous increase in outcrossing and recombination potential, due in part to selective acquisition (under virus pressure) of MAT-1 and vic loci from the resident O. ulmi and in part to SSAM × SSNU hybridisation. Overt virus infection in the 2008 samples was low (~4%), diagnostic SSAM and SSNU cu and col1 loci were recombinant, and no isolates exhib-ited a parental SSAM or SSNU colony pattern. At both sites, mean growth rate and mean patho-genicity to 3–5 m clonal elm were high SSAM-like, indicating sustained directional selection for these characters, though at Orvieto growth rate was slower. The once frequent SSNU-specific up-mut colony dimorphism was largely eliminated at both sites. Perithecia formed by Limburg isolates were mainly an extreme, long-necked SSNU-like form, consistent with transgressive segregation resulting from mismatch of SSAM and SSNU developmental loci. Orvieto isolates produced more parental-like perithecia, suggesting the extreme phenotypes may have been se-lected against. The novel phenotypes in the swarms are remodelling O. novo-ulmi in Europe. Locally adapted genotypes may emerge.

Monitoring the state of St. Petersburg tree plantations: modern and traditional approaches

Предлагается использование системы Tree Talker мониторинга в сочетании с традиционными методами фитопатологического и энтомологического мониторинга для обеспечения своевременного обнаружения изменения состояния насаждений Санкт-Петербурга и выявления ключевых факторов экологического стресса. Разнообразная ведомственная принадлежность и широкий видовой состав насаждений города, а также наличие многочисленных экологических факторов, негативно влияющих на состояние деревьев, появление инвазионных патогенов и вредителей создают плохо прогнозируемые ситуации. Появление инвазионных вредителей и патогенов, таких как возбудитель голландской болезни гриб-аскомицет Ophiostoma novo-ulmi и его распространители – короеды-заболонники, ещё один аскомицет, гриб Hymenoscyphus fraxineus и ясеневая изумрудная узкотелая златка Agrilus planipennis, привело к массовой гибели вязов и ясеней, в том числе из-за несвоевременного обнаружения этих патогенов и вредителей. В условиях разобщенности системы управления городскими насаждениями из-за разной ведомственной принадлежности, мозаичного расположения, видового разнообразия и специфичности видового состава древесных растений, а также требований к оперативному принятию решений, использование Tree Talker технологий становится весьма перспективным. Эти технологии позволяют обеспечить оперативное получение, передачу и анализ данных по суточной и сезонной динамике физиологических параметров, устойчивости деревьев к ветровым нагрузкам с учётом породы, возраста и размеров. Интегрирование полученных данных позволяет оценить эффективность насаждений в целом по созданию микроклимата, динамике отклонений по вертикали, эффективности санитарно-оздоровительных мероприятий. В итоге, получение оперативной информации позволяет своевременно обнаружить неблагоприятные изменения как у отдельных деревьев, так и в структуре насаждений и провести анализ причин этих изменений, в особенности в отношении появления биологических угроз насаждения – распространению опасных патогенов и размножению вредителей. It is proposed to use the Tree Talker monitoring system in combination with traditional methods of phytopathological and entomological monitoring to ensure timely detection of changes in the state of plantations in St. Petersburg and identify key factors of environmental stress. Diverse departmental affiliation and a wide species composition of the city's plantations, as well as the presence of numerous environmental factors that negatively affect the condition of trees, the appearance of invasive pathogens and pests create poorly predictable situations. The emergence of invasive pests and pathogens, such as the causative agent of the Dutch elm disease, the Ascomycete fungi Ophiostoma novo-ulmi and its spreaders – the sapwood bark beetles, another Ascomycete, the Hymenoscyphus fraxineus fungus, and the Emerald ash borer Agrilus planipennis in including due to late detection of these pathogens and pests. In the context of the disunity of the management system of urban plantings due to different departmental affiliation, mosaic location, species diversity and specificity of the species composition of woody plants, as well as requirements for prompt decision-making, the use of Tree Talker technologies becomes very promising. These technologies allow for the prompt receipt, transmission and analysis of data on the daily and seasonal dynamics of physiological parameters, the resistance of trees to wind loads, taking into account the species, age and size. Integration of the obtained data allows us to evaluate the effectiveness of plantings in general in terms of creating a microclimate, the dynamics of vertical deviations, and the effectiveness of sanitary and recreational activities. As a result, obtaining operational information allows us to timely detect unfavorable changes both in individual trees and in the structure of plantings and to analyze the causes of these changes, especially in relation to the emergence of biological threats to the plantation – the spread of dangerous pathogens and the reproduction of pests.