Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high ...importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.
Cryphonectria parasitica was detected for the first time in the United Kingdom in 2011. A 2017–2018 survey detected the disease at different sites in Berkshire, Derbyshire, Devon, Dorset and London, ...while the present study comprises the results of the 2019–2020 survey with new findings and additional sites in the United Kingdom (Berkshire, Buckinghamshire, Cornwall, Derbyshire, Devon, London, West Sussex) and in Jersey, reflecting the progressive detection of more infected trees. A total of 189 samples were collected from 52 sites, and 123 samples tested positive both by quantitative real‐time PCR and/or isolation from 43 sites. A total of 115 isolates were tested for mating type, vegetative compatibility group (VCG) and Cryphonectria hypovirus 1 (CHV‐1). Twelve VCGs were identified, with four of them being first records in United Kingdom. The highest diversity of VCGs was detected in Devon followed by West Sussex while London and Derbyshire presented the lowest. Both mating types were detected (41% MAT‐1 and 59% MAT‐2), and no heterokaryons were detected. Perithecia of C. parasitica were not observed at any site during this survey. CHV‐1 was detected in three isolates in very low concentration from three different locations in London and was always the unmutated subtype I haplotype E‐5. A greater diversity of VCGs at outbreak sites compared with previous surveys, combined with their scattered distribution and the slow spread of the pathogen, supports the hypothesis that this disease has been introduced through imports over time from Europe.
Twelve VCGs were identified, the highest diversity in Devon followed by West Sussex. Both mating types were present, but no heterokaryons or perithecia were observed. CHV‐1 was found infecting three isolates in low concentration.
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Some mycoviruses such as Cryphonectria hypovirus 1 (CHV-1) have a potential application for biocontrol of Cryphonectria parasitica, the causal agent of chestnut blight, but little is ...known about the factors that regulate the fungus-virus interaction, especially when the concentration of the virus is low as it is usual in the wider environment in England. In this study, four CHV-1 infected isolate types of C. parasitica were tested for parameters relating to hypovirulence by inoculating onto potato dextrose agar (PDA), apples, and sweet chestnut branch fragments. Isolate L-6 (CHV-1 subtype I, haplotype E-5), together with the positive control isolate M784 (same haplotype), presented the highest hypovirulence values. The lowest hypovirulence was observed with isolate M784 previously kept in the cold at 8 °C, followed by isolate Db-1 (same haplotype but naturally mutated). The differences in hypovirulence were much more pronounced when inoculations took place using mycelial plugs taken from the edge of plate colonies as opposed to the centre of a colony. The same strains were used to further evaluate the effects of the growth medium (PDA or plant material), optimum and cold temperatures, and mycelium age (centre or edge plugs) on the respective virus concentration. Virus RNA was extracted and amplified by RT-PCR and the concentration of the CHV-1 DNA band (394 bp) measured. Virus concentration decreased when its host was growing on PDA when compared to its host growing on plant material. Virus concentration was reduced further if held in the cold temperature for 7 days, and the virus concentration decreased even further after an additional incubation period at 25 °C. Virus isolate L-6, sub-cultured from the colony centre, was the only one that recovered to its original concentration following incubation for 7 days on sweet chestnut wood fragments. The concentration of the virus was more stable in the centre of the plates than that found at the edge of a plate colony. Together with the expected 394 bp CHV-1 amplicon, another 90 bp band was occasionally simultaneously amplified using the same RT-PCR procedure. This band was quantified, and sequenced, finding no homologs. It is first time this satellite amplicon has been described and its concentration related with hypovirulence attributes of the virus infected C. parasitica, such as reduced fungal growth, and positive correlation with donor and recipient CHV-1 band load in donor-recipient transmission experiments. We discuss our findings in terms of previous parameters related to hypovirulence.
Root rot, caused by Phytophthora cinnamomi is the most important disease of avocado, but few studies have determined whether other Phytophthora or oomycete species are involved in crop decline. ...Avocado orchards in the Canary Islands were surveyed for the presence of Phytophthora and Phytophthora-like oomycetes. Isolates obtained were identified morphologically and by sequence analysis of their internal transcribed spacer (ITS) regions, and their pathogenicity was tested by root and stem inoculation of avocado seedlings. Phytophthora species were isolated in 41 of 99 orchards sampled, and 10% of orchards were infected with more than one species. The species most frequently isolated was P. cinnamomi, which was detected in 26 orchards. In addition, P. multivora (ten orchards), P. niederhauserii (four orchards), P. nicotianae (four orchards), P. palmivora (one orchard) and Phytopythium vexans (20 orchards) were isolated. Phytophthora nicotianae and P. palmivora have been previously reported as pathogens of avocado, but P. niederhauserii, P. multivora and Pp. vexans are reported for the first time to be associated with this host. Phytophthora niederhauserii was the most virulent of these species. It was isolated from declining trees, and root rot severity was comparable to that caused by P. cinnamomi in two independent pathogenicity tests. In addition, P. niederhauserii caused cankers after stem inoculation. The pathogenicity results for P. multivora and Pp. vexans varied depending on isolates and pathogenicity tests. This study increases the knowledge of oomycetes associated with avocado, highlighting the potential threat posed by P. niederhauserii to this important fruit crop.
Plant pathogens are introduced to new geographical regions ever more frequently as global connectivity increases. Predicting the threat they pose to plant health can be difficult without in‐depth ...knowledge of behaviour, distribution and spread. Here, we evaluate the potential for using biological traits and phylogeny to predict global threats from emerging pathogens.
We use a species‐level trait database and phylogeny for 179 Phytophthora species: oomycete pathogens impacting natural, agricultural, horticultural and forestry settings. We compile host and distribution reports for Phytophthora species across 178 countries and evaluate the power of traits, phylogeny and time since description (reflecting species‐level knowledge) to explain and predict their international transport, maximum latitude and host breadth using Bayesian phylogenetic generalised linear mixed models.
In the best‐performing models, traits, phylogeny and time since description together explained up to 90%, 97% and 87% of variance in number of countries reached, latitudinal limits and host range, respectively. Traits and phylogeny together explained up to 26%, 41% and 34% of variance in the number of countries reached, maximum latitude and host plant families affected, respectively, but time since description had the strongest effect.
Root‐attacking species were reported in more countries, and on more host plant families than foliar‐attacking species. Host generalist pathogens had thicker‐walled resting structures (stress‐tolerant oospores) and faster growth rates at their optima. Cold‐tolerant species are reported in more countries and at higher latitudes, though more accurate interspecific empirical data are needed to confirm this finding.
Policy implications. We evaluate the potential of an evolutionary trait‐based framework to support horizon‐scanning approaches for identifying pathogens with greater potential for global‐scale impacts. Potential future threats from Phytophthora include Phytophthora x heterohybrida, P. lactucae, P. glovera, P. x incrassata, P. amnicola and P. aquimorbida, which are recently described, possibly under‐reported species, with similar traits and/or phylogenetic proximity to other high‐impact species. Priority traits to measure for emerging species may be thermal minima, oospore wall index and growth rate at optimum temperature. Trait‐based horizon‐scanning approaches would benefit from the development of international and cross‐sectoral collaborations to deliver centralised databases incorporating pathogen distributions, traits and phylogeny.
We evaluate the potential of an evolutionary trait‐based framework to support horizon‐scanning approaches for identifying pathogens with greater potential for global‐scale impacts. Potential future threats from Phytophthora include Phytophthora x heterohybrida, P. lactucae, P. glovera, P. x incrassata, P. amnicola and P. aquimorbida, which are recently described, possibly under‐reported species, with similar traits and/or phylogenetic proximity to other high‐impact species. Priority traits to measure for emerging species may be thermal minima, oospore wall index and growth rate at optimum temperature. Trait‐based horizon‐scanning approaches would benefit from the development of international and cross‐sectoral collaborations to deliver centralised databases incorporating pathogen distributions, traits and phylogeny.
As global plant trade expands, tree disease epidemics caused by pathogen introductions are increasing. Since ca 2000, the introduced oomycete
has caused devastating epidemics in Europe and North ...America, spreading as four ancient clonal lineages, each of a single mating type, suggesting different geographical origins. We surveyed laurosilva forests for
around Fansipan mountain on the Vietnam-China border and on Shikoku and Kyushu islands, southwest Japan. The surveys yielded 71
isolates which we assigned to eight new lineages, IC1 to IC5 from Vietnam and NP1 to NP3 from Japan, based on differences in colony characteristics, gene x environment responses and multigene phylogeny. Molecular phylogenetic trees and networks revealed the eight Asian lineages were dispersed across the topology of the introduced European and North American lineages. The deepest node within
, the divergence of lineages NP1 and NP2, was estimated at 0.5 to 1.6 Myr. The Asian lineages were each of a single mating type, and at some locations, lineages of "opposite" mating type were present, suggesting opportunities for inter-lineage recombination. Based on the high level of phenotypic and phylogenetic diversity in the sample populations, the coalescence results and the absence of overt host symptoms, we conclude that
comprises many anciently divergent lineages native to the laurosilva forests between eastern Indochina and Japan.
In 2016 and 2017, surveys of Phytophthora diversity were performed in 25 natural and semi-natural forest stands and 16 rivers in temperate and subtropical montane and tropical lowland regions of ...Vietnam. Using baiting assays from soil samples and rivers and direct isolations from naturally fallen leaves, 13 described species, five informally designated taxa and 21 previously unknown taxa of Phytophthora were isolated from 58 of the 91 soil samples (63.7%) taken from the rhizosphere of 52 of the 64 woody plant species sampled (81.3%) in 20 forest stands (83.7%), and from all rivers: P. capensis, P. citricola VII, VIII, IX, X and XI, P. sp. botryosa-like 2, P. sp. meadii-like 1 and 2, P. sp. tropicalis-like 2 and P. sp. multivesiculata-like 1 from Phytophthora major phylogenetic Clade 2; P. castaneae and P. heveae from Clade 5; P. chlamydospora, P. gregata, P. sp. bitahaiensis-like and P. sp. sylvatica-like 1, 2 and 3 from Clade 6; P. cinnamomi (Pc), P. parvispora, P. attenuata, P. sp. attenuata-like 1, 2 and 3 and P. ×heterohybrida from Clade 7; P. drechsleri, P. pseudocryptogea, P. ramorum (Pr) and P. sp. kelmania from Clade 8, P. macrochlamydospora, P. sp. ×insolita-like, P. sp. ×kunnunara-like, P. sp. ×virginiana-like s.l. and three new taxa, P. sp. quininea-like, P. sp. ×Grenada 3-like and P. sp. ×Peru 4-like, from Clade 9; and P. sp. gallica-like 1 and 2 from Clade 10. The A1 and A2 mating types of both Pc and Pr co-occurred. The A2 mating type of Pc was associated with severe dieback of montane forests in northern Vietnam. Most other Phytophthora species, including Pr, were not associated with obvious disease symptoms. It is concluded that (1) Vietnam is within the center of origin of most Phytophthora taxa found including Pc and Pr, and (2) Phytophthora clades 2, 5, 6, 7, 8, 9, and 10 are native to Indochina.
The plant nursery industry has become an ideal reservoir for Phytophthora species and other soilborne pathogens. In this context, isolation from tissues and soil of ornamental and forest plants from ...nurseries in four regions of Spain was carried out. A high diversity of Phytophthora species was confirmed. Fourteen Phytophthora phylotypes (P. cactorum, P. cambivora, P. cinnamomi, P. citrophthora, P. crassamura, P. gonapodyides, P. hedraiandra, P. nicotianae, P. niederhauserii, P. palmivora, P. plurivora, P. pseudocryptogea, P. sansomeana, and Phytophthora sp. tropicalis-like 2) were isolated from over 500 plant samples of 22 species in 19 plant genera. Nine species were detected in water sources, two of them (P. bilorbang and P. lacustris) exclusively from water samples. P. crassamura was detected for the first time in Spain. This is the first time P. pseudocryptogea is isolated from Chamaecyparis lawsoniana and Yucca rostrata in Spain.
Biological control of
fungus, causal agent of chestnut blight, by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe and some parts ...of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV-1) type species of the
family. To efficiently provide biocontrol, the virus must be able to induce hypovirulence in its fungal host in chestnut trees. Here, two different CHV-1 subtype I virus strains (E-5 and L-18), gained by transmissions, were tested for their hypovirulence induction, biocontrol potential, and transmission between vegetatively compatible (VCG) and incompatible fungal isolate groups in sweet chestnut seedlings and branches. Both strains of CHV-1 showed great biocontrol potential and could protect trees by efficiently transmitting CHV-1 by hyphal anastomosis between fungal isolates of the same VCG and converting virulent to hypovirulent cankers. The hypovirulent effect was positively correlated with the virus concentration, tested by four different reverse-transcription PCRs, two end-point and two real-time methods, one of which represents a newly developed real-time PCR for the detection and quantification of CHV-1.
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