There are several highly damaging Phytophthora species pathogenic to forest trees, many of which have been spread beyond their native range by the international trade of live plants and infested materials. Such introductions can be reduced through the development of better tools capable of the early, rapid, and high-throughput detection of contaminated plants. This study utilized a volatilomics approach (solid-phase microextraction coupled to gas chromatography-mass spectrometry) to differentiate between several Phytophthora species in culture and discriminate between healthy and Phytophthora-inoculated European beech and pedunculate oak trees. We tentatively identified 14 compounds that could differentiate eight Phytophthora species from each other in vitro. All of the Phytophthora species examined, except Phytophthora cambivora, uniquely produced at least one compound not observed in the other species; however, most detected compounds were shared between multiple species. Phytophthora polonica had the most unique compounds and was the least similar of all the species examined. The inoculated seedlings had qualitatively different volatile profiles and could be distinguished from the healthy controls by the presence of isokaurene, anisole, and a mix of three unknown compounds. This study supports the notion that volatiles are suitable for screening plant material, detecting tree pathogens, and differentiating between healthy and diseased material.

Western redcedar (Thuja plicata Donn ex D. Don) is one of the most important commercial tree species in British Columbia, generates more than $1 billion in economic activity annually and about 8-10 million trees are planted in reforestation efforts (Gregory et al. 2018). It has been selected as the provincial tree of British Columbia (BC) because of its tremendous economic, ecoogical and cultural value. However, foliar diseases such as leaf blights have serious impact on redcedar growth and may cause significant loss of tree volume (Russell, 2007). Our 2014 - 2015 surveys of western redcedar forests in coastal areas of BC indicated high incidence of a distinctive type of blight. We observed the incidence of this disease on more than 80% of western redcedar (approximately 493) trees from late May to early December. Early symptoms appeared as circular to oval, brownish to black spots (2-3 mm), 1-5 spots per branch tip, scattered at the tip margins. Sequentially, the spots enlarged and developed into necrotic lesions on both young and old leaves. More than 50 symptomatic leaves from 10 different trees were collected and rinsed in distilled water then surface-sterilized with three times washing in Tween 20 (%5 solution) for 2 minutes (each time) and %70 ethanol for 30 second (3 times repeat). Tissues from under lesions were placed on MEA (Malt Extract Agar; Phyto Tech® labs-Product ID: M498) and PDA (Potato Dextrose Agar; Phyto Tech® Labs-Product ID: P772). The plates were incubated at 21°C in the dark. They developed distinct dull white to brown, cottony colonies with each black acervuli approximately 450-500µm. The isolates produced fusiform conidia with four cells. They didn\'t have any distinct color. The conidiophore size was approximately 23-24 x 2-3 µm with mostly hyaline to light brown color, branched and conidiogenous was hyaline and not branched and simple. The spore size was approximately 15-20μm by 7-10μm with three transverse septa and endogenous papillae with hyaline apical appendages. Next, we collected spores and replated them on fresh MEA media culture and placed back in the incubator to produce pure cultures. We studied conidia from leaves of trees mentioned above using light and electron microscopy using Hitachi S-3500N Scanning Electron Microscope (Noshad et al. 2023). After morphological study, further identification to the species level conducted using Zambounis and Wenneker\'s approach (Zambounis 2019; Wenneker,2017). Genomic DNA from two single-spore isolates were isolated and sequenced. Sequences of ITS (Internal Transcribed Spacer) region amplified using primers ITS1/ITS4 and sequenced. Final sequences were deposited in Genbank and published (accession numbers OP086244 and OP086251). Blast analysis of these sequences showed 99% and 99% resemblances with T. angustata sequence (Sutton 1980). To verify its pathogenicity, we performed a comprehensive pathogenicity test to fulfill Koch\'s postulates. We collected their distinctive spores in an aseptic environment and standardized them (5000/ml) using a haemocytometer. Then we inoculated 100 western redcedar seedlings (three years old) by injecting standardized spore suspension solution (inoculum) using ultra-fine 0.3ml, 31G, 8mm syringes (approximately 0.1ml per inoculation site). Ten positive control seedlings were inoculated with distilled water and ten negative control seedlings were not inoculated at all. All inoculated (experimental) seedlings demonstrated same symptoms (black spots and characteristic spores) after eight weeks. None of the control seedlings showed any similar symptoms. In the next stage, we isolated and cultured spores from inoculated seedlings and studied them. The identity of reisolates confirmed using DNA sequencing. We used these spores for our next set of disease screening which was successful again. We identified Truncatella angustata (Pers.) Hughes as the causal agent for shoot-tip blight (STB) on western redcedar by examining morphological and molecular characteristics of the pathogen. This is the first report of T. angustata as a primary pathogen on western redcedar in British Columbia, Canada.

Bacterial canker is a major disease of stone fruits and is a critical limiting factor to sweet cherry (Prunus avium) production worldwide. One important strategy for disease control is the development of resistant varieties. Partial varietal resistance in sweet cherry is discernible using shoot or whole tree inoculations; however, these quantitative differences in resistance are not evident in detached leaf assays. To identify novel sources of resistance to canker, we used a rapid leaf pathogenicity test to screen a range of wild cherry, ornamental Prunus species and sweet cherry × ornamental cherry hybrids with the canker pathogens, Pseudomonas syringae pvs syringae, morsprunorum races 1 and 2, and avii. Several Prunus accessions exhibited limited symptom development following inoculation with each of the pathogens, and this resistance extended to 16 P. syringae strains pathogenic on sweet cherry and plum. Resistance was associated with reduced bacterial multiplication after inoculation, a phenotype similar to that of commercial sweet cherry towards nonhost strains of P. syringae. Progeny resulting from a cross of a resistant ornamental species Prunus incisa with susceptible sweet cherry (P. avium) exhibited resistance indicating it is an inherited trait. Identification of accessions with resistance to the major bacterial canker pathogens is the first step towards characterizing the underlying genetic mechanisms of resistance and introducing these traits into commercial germplasm.

The genus Calonectria includes pathogens of various agricultural, horticultural, and forestry crops. Species of Calonectria are commonly collected from soils, fruits, leaves, stems, and roots. Some species of Calonectria isolated from soils are considered as important plant pathogens. Understanding the species diversity and distribution characteristics of Calonectria species in different soil layers will help us to clarify their long-term potential harm to plants and their patterns of dissemination. To our knowledge, no systematic research has been conducted concerning the species diversity and distribution characteristics of Calonectria in different soil layers. In this study, 1000 soil samples were collected from five soil layers (0-20, 20-40, 40-60, 60-80, and 80-100 cm) at 100 sampling points in one 15-year-old Eucalyptus urophylla hybrid plantation in southern China. A total of 1037 isolates of Calonectria present in all five soil layers were obtained from 93 of 100 sampling points. The 1037 isolates were identified based on DNA sequence comparisons of the translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) gene regions, as well as the combination of morphological characteristics. These isolates were identified as C. hongkongensis (665 isolates; 64.1%), C. aconidialis (250 isolates; 24.1%), C. kyotensis (58 isolates; 5.6%), C. ilicicola (47 isolates; 4.5%), C. chinensis (2 isolates; 0.2%), and C. orientalis (15 isolates; 1.5%). With the exception of C. orientalis, which resides in the C. brassicae species complex, the other five species belonged to the C. kyotensis species complex. The results showed that the number of sampling points that yielded Calonectria and the number (and percentage) of Calonectria isolates obtained decreased with increasing depth of the soil. More than 84% of the isolates were obtained from the 0-20 and 20-40 cm soil layers. The deeper soil layers had comparatively lower numbers but still harbored a considerable number of Calonectria. The diversity of five species in the C. kyotensis species complex decreased with increasing soil depth. The genotypes of isolates in each Calonectria species were determined by tef1 and tub2 gene sequences. For each species in the C. kyotensis species complex, in most cases, the number of genotypes decreased with increasing soil depth. The 0-20 cm soil layer contained all of the genotypes of each species. To our knowledge, this study presents the first report of C. orientalis isolated in China. This species was isolated from the 40-60 and 60-80 cm soil layers at only one sampling point, and only one genotype was present. This study has enhanced our understanding of the species diversity and distribution characteristics of Calonectria in different soil layers.

Phytophthora species cause some of the most serious diseases of trees and threaten forests in many parts of the world. Despite the generation of genome sequence assemblies for over 10 tree-pathogenic Phytophthora species and improved detection methods, there are many gaps in our knowledge of how these pathogens interact with their hosts. To facilitate cell biology studies of the infection cycle we examined whether the tree pathogen Phytophthora kernoviae could infect the model plant Nicotiana benthamiana. We transformed P. kernoviae to express green fluorescent protein (GFP) and demonstrated that it forms haustoria within infected N. benthamiana cells. Haustoria were also formed in infected cells of natural hosts, Rhododendron ponticum and European beech (Fagus sylvatica). We analysed the transcriptome of P. kernoviae in cultured mycelia, spores, and during infection of N. benthamiana, and detected 12,559 transcripts. Of these, 1,052 were predicted to encode secreted proteins, some of which may function as effectors to facilitate disease development. From these, we identified 87 expressed candidate RXLR (Arg-any amino acid-Leu-Arg) effectors. We transiently expressed 12 of these as GFP fusions in N. benthamiana leaves and demonstrated that nine significantly enhanced P. kernoviae disease progression and diversely localized to the cytoplasm, nucleus, nucleolus, and plasma membrane. Our results show that N. benthamiana can be used as a model host plant for studying this tree pathogen, and that the interaction likely involves suppression of host immune responses by RXLR effectors. These results establish a platform to expand the understanding of Phytophthora tree diseases.

Many Calonectria species are causal agents of diseases on several forestry, agricultural and horticultural crops. Calonectria leaf blight is one of the most important diseases associated with Eucalyptus plantations and nurseries in Asia and South America. Recently, symptoms of leaf rot and leaf blight caused by Calonectria species were observed in a one-year-old Eucalyptus experimental plantation in GuangXi Province, southern China. To better understand the species diversity, mating strategy and pathogenicity of Calonectria species isolated from diseased tissues and soils, diseased leaves and soils under the trees from ten Eucalyptus urophylla hybrid genotypes were collected. Three hundred and sixty-eight Calonectria isolates were obtained from diseased Eucalyptus leaves and soils under these trees, and 245 representative isolates were selected based on the sampling substrates and Eucalyptus genotypes and identified by DNA sequence analyses based on the translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA) and histone H3 (his3) gene regions, as well as a combination of morphological characteristics. These isolates were identified as Calonectria hongkongensis (50.2%), C. pseudoreteaudii (47.4%), C. aconidialis (1.6%), C. reteaudii (0.4%) and C. auriculiformis (0.4%). This is the first report of C. reteaudii and C. auriculiformis occurrence in China. Calonectria pseudoreteaudii was isolated from both Eucalyptus diseased leaves and soils; the other four species were only obtained from soils. MAT1-1-1 and MAT1-2-1 gene amplification and mating type assignment results showed that C. pseudoreteaudii is heterothallic and an asexual cycle represents the primary reproductive mode, C. reteaudii and C. auriculiformis are likely to be heterothallic and C. hongkongensis and C. aconidialis are homothallic. Based on the genetic diversity comparisons for C. pseudoreteaudii isolates from diseased leaves and soils, we hypothesize that C. pseudoreteaudii in soils was spread from diseased leaves. Both the mycelia plug and conidia suspension inoculations indicated that all five Calonectria species were pathogenic to the two Eucalyptus genotypes tested and the tolerance of the two genotypes differed. It is necessary to understand the ecological niche and epidemiological characteristics of these Calonectria species and to select disease resistant Eucalyptus genotypes in southern China in the future.

Eucalyptus (Myrtaceae, Myrtales) trees are widely cultivated for commercial purposes worldwide. Calonectria leaf blight is one of the most prominent diseases associated with Eucalyptus trees grown in plantations in Asia and South America. Recently, symptoms of leaf blight, shoot blight, tree death, and seedling rot caused by Calonectria species have been observed in commercial Eucalyptus plantations and nurseries in Leizhou Peninsula, which is one of the most densely Eucalyptus-planted areas in southern China. Disease samples were collected from 10 Eucalyptus species and a number of Eucalyptus grandis, E. tereticornis, and E. urophylla hybrid genotypes that were planted on plantations at 13 sites and one experimental nursery. A total of 773 isolates of Calonectria were obtained from 683 plantation trees and nursery seedlings. Fifty-five representative isolates from all the surveyed sites and Eucalyptus species/genotypes were selected for molecular identification. These 55 isolates were identified by DNA sequence analyses based on the calmodulin (cmdA), histone H3 (his3), translation elongation factor 1-alpha (tef1), and β-tubulin (tub2) gene regions, as well as a combination of morphological characteristics. The results indicated that these 55 isolates present one single species, Calonectria pentaseptata. Determined by sequences of cmdA, his3, tef1, and tub2 gene regions, only two genotypes were identified among the 55 representative isolates; 54 of these isolates share the same genotype, suggesting that the genetic diversity of Ca. pentaseptata collected during this study was relatively low. A growth study indicated that Ca. pentaseptata is a high-temperature species. The mating test results suggested that Ca. pentaseptata is heterothallic or lacks the ability to recombine to produce fertile progeny. Inoculation results showed that Ca. pentaseptata causes leaf blight and stem rot, resulting in tree death of the two widely planted Eucalyptus genotypes in southern China, and that the two genotypes differ significantly in their susceptibility to infection by Ca. pentaseptata. A selection program to develop Eucalyptus planting stocks with high levels of resistance to Calonectria leaf blight in China during the long-term should be urgently initiated.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pitch canker, caused by Gibberella circinata (anamorph = Fusarium circinatum), causes canopy dieback and mortality in susceptible pine species in many parts of the world. Pitch canker is most problematic in areas with a relatively warm climate, suggesting a possible limitation on disease development imposed by low temperatures. To test this hypothesis, the effect of temperature on radial growth was examined in isolates of G. circinata of diverse geographic origin. All isolates grew most rapidly at 25°C and progressively more slowly at 20, 15, and 10°C. Spore germination occurred most rapidly at 20°C and was slowest at 10°C. To determine if the time required for spore germination might influence the likelihood of infection, the duration of wound susceptibility was examined by inoculating branches of susceptible Monterey pines (Pinus radiata). In each of six field trials, branches were wounded and then inoculated immediately or at 2, 6, or 9 days after wounding. The results indicated that wounds inoculated immediately became infected at a significantly higher rate than those inoculated 2 days later. Thus, if low temperatures extend the time required for germination beyond this period, a reduced infection frequency would be expected. Such a limiting effect of temperature could help to explain the current distribution of pitch canker.

To manage emerging forest diseases and prevent their occurrence in the future, it is essential to determine the origin(s) of the pathogens involved and identify the management practices that have ultimately caused disease problems. One such practice is the widespread planting of exotic tree species within the range of related native taxa. This can lead to emerging forest disease both by facilitating introduction of exotic pathogens and by providing susceptible hosts on which epidemics of native pathogens can develop. We used microsatellite markers to determine the origins of the pathogen Dothistroma septosporum responsible for the current outbreak of Dothistroma needle blight (DNB) on native Caledonian Scots pine (Pinus sylvestris) populations in Scotland and evaluated the role played by widespread planting of two exotic pine species in the development of the disease outbreak. We distinguished three races of D. septosporum in Scotland, one of low genetic diversity associated with introduced lodgepole pine (Pinus contorta), one of high diversity probably derived from the DNB epidemic on introduced Corsican pine (Pinus nigra subsp. laricio) in England and a third of intermediate diversity apparently endemic on Caledonian Scots pine. These races differed for both growth rate and exudate production in culture. Planting of exotic pine stands in the UK appears to have facilitated the introduction of two exotic races of D. septosporum into Scotland which now pose a threat to native Caledonian pines both directly and through potential hybridization and introgression with the endemic race. Our results indicate that both removal of exotic species from the vicinity of Caledonian pine populations and restriction of movement of planting material are required to minimize the impact of the current DNB outbreak. They also demonstrate that planting exotic species that are related to native species reduces rather than enhances the resilience of forests to pathogens.

Bark beetles form multipartite symbiotic associations with blue stain fungi (Ophiostomatales, Ascomycota). These fungal symbionts play an important role during the beetle\'s life cycle by providing nutritional supplementation, overcoming tree defences and modifying host tissues to favour brood development. The maintenance of stable multipartite symbioses with seemingly less competitive symbionts in similar habitats is of fundamental interest to ecology and evolution. We tested the hypothesis that the coexistence of three fungal species associated with the mountain pine beetle is the result of niche partitioning and adaptive radiation using SNP genotyping coupled with genotype-environment association analysis and phenotypic characterization of growth rate under different temperatures. We found that genetic variation and population structure within each species is best explained by distinct spatial and environmental variables. We observed both common (temperature seasonality and the host species) and distinct (drought, cold stress, precipitation) environmental and spatial factors that shaped the genomes of these fungi resulting in contrasting outcomes. Phenotypic intraspecific variations in Grosmannia clavigera and Leptographium longiclavatum, together with high heritability, suggest potential for adaptive selection in these species. By contrast, Ophiostoma montium displayed narrower intraspecific variation but greater tolerance to extreme high temperatures. Our study highlights unique phenotypic and genotypic characteristics in these symbionts that are consistent with our hypothesis. By maintaining this multipartite relationship, the bark beetles have a greater likelihood of obtaining the benefits afforded by the fungi and reduce the risk of being left aposymbiotic. Complementarity among species could facilitate colonization of new habitats and survival under adverse conditions.