Isolation and pathogenicity of Phytophthora species from declining Rubus anglocandicans

Rubus anglocandicans is the most widespread and abundant blackberry species within the European blackberry (Rubus fruticosus) aggregate in Western Australia (WA). European blackberry is also one of the 32 Weeds of National Significance in Australia. A disease recorded as ‘blackberry decline’ was first observed in some blackberry sites in WA in 2006. A disease survey was conducted in the Manjimup‐Pemberton region along the Warren and Donnelly River catchments in WA between 2010 and 2012. Phytophthora amnicola, P. bilorbang, P. cryptogea, P. inundata, P. litoralis, P. multivora, P. taxon personii, P. thermophila and a P. thermophila × amnicola hybrid were recovered from declining and adjacent decline‐free sites, as well as from streams and rivers. Phytophthora cinnamomi was isolated from dying Banksia and Eucalyptus species from two non‐decline sites. Of these species, P. bilorbang and P. cryptogea were more pathogenic than the others in under‐bark inoculations using excised stems (primocanes), in planta primocane inoculations in blackberry growing wild in native forest stands, and in glasshouse pot trials. It was concluded that blackberry decline is a complex syndrome and Phytophthora species, in particular P. bilorbang and P. cryptogea, together with temporary inundation, are major biotic and abiotic factors contributing to blackberry decline.     

Two novel and potentially endemic species of Phytophthora associated with episodic dieback of Kwongan vegetation in the south‐west of Western Australia

Two novel homothallic species of Phytophthora causing dieback of Kwongan vegetation in south‐west Western Australia are described here as Phytophthora arenaria sp. nov. and Phytophthora constricta sp. nov. DNA sequencing of the ITS rDNA and cox1 gene confirmed that P. arenaria and P. constricta are unique species residing in ITS clades 4 and 9, respectively. Phytophthora arenaria has been isolated from vegetation occurring on the northern sandplains which are warmer and drier than the southern sandplains from which P. constricta has been predominantly isolated, and both species appear morphologically and physiologically well adapted to the ecosystems in which they occur. Both species have been associated mainly with dead and dying Banksia species and the pathogenicity of both P. arenaria and P. constricta to Banksia attenuata was confirmed in this study. The combination of unique DNA sequences, including considerable variation in cox1 sequence data, thick oospore walls and physiological characteristics that appear to be adaptations favouring survival in the harsh Kwongan ecosystem suggest that these species may be endemic to Western Australia.     

Phomopsis husk rot of macadamia in Australia and South Africa caused by novel Diaporthe species

Phomopsis husk rot (PHR) in macadamia is a disease of economic importance in major commercial production areas in Australia and South Africa. Effective control of PHR is hindered by limited knowledge about its aetiology and epidemiology. The diversity and pathogenicity of more than 50 isolates of Diaporthe associated with PHR in macadamia orchards in Australia and South Africa was assessed. Multilocus phylogenetic analyses of DNA sequences of the ITS, tef1α, and tub2 gene loci revealed four novel clades that are described as Diaporthe australiana sp. nov., D. drenthii sp. nov., D. macadamiae sp. nov., and D. searlei sp. nov. Pathogenicity tests with representative isolates found that all four species caused PHR of varying severity between and within species, as well as between the two macadamia cultivars HAES 344 and HAES 816. The Australian species, D. australiana, was the most aggressive species compared with the three South African species. This study improves our understanding of the aetiology of PHR in macadamia and paves the way for more effective disease management.     

The morphology,behaviour and molecular phylogeny of Phytophthora taxon Salixsoil and its redesignation as Phytophthora lacustris sp. nov.

Since its first isolation from Salix roots in 1972, isolates of a sexually sterile Phytophthora species have been obtained frequently from wet or riparian habitats worldwide and have also been isolated from roots of Alnus and Prunus spp. Although originally assigned to Phytophthora gonapodyides on morphological grounds, it was recognized that these isolates, informally named P. taxon Salixsoil, might represent a separate lineage within ITS Clade 6. Based on phylogenetic analyses and comparisons of morphology, growth‐temperature relationships and pathogenicity, this taxon is formally described here as Phytophthora lacustris sp. nov. Isolates of P. lacustris form a clearly resolved cluster in both ITS and mitochondrial cox1 phylogenies, basal to most other Clade 6 taxa. Phytophthora lacustris shares several unusual behavioural properties with other aquatic Clade 6 species, such as sexual sterility and tolerance of high temperatures, that have been suggested as adaptations to riparian conditions. It appears to be widespread in Europe and has also been detected in Australia, New Zealand and the USA. It was shown to be weakly or moderately aggressive on inoculation to Alnus, Prunus and Salix. The extent of P. lacustris’ activity as a saprotroph in plant debris in water and as an opportunistic pathogen in riparian habitats needs further investigation. Its pathogenic potential to cultivated fruit trees also deserves attention because P. lacustris has apparently been introduced into the nursery trade.     

Notes on predacious mites of greece,with a description of five new species (mesostigmata: phytoseiidae)

The following 14 species of phytoseiid mites were recorded on various plants:Typhlodromus kerkirae sp. nov.,T. psyllakisi sp. nov.,T. athenas sp. nov.,T. aff.recki Wainstein,T. cryptus Athias-Henriot,T. atticus sp. nov.,T. longipalpus sp. nov.,T. tiliaruml Oudemans,Seiulus isotrichus Athias-Henriot,Amblyseius aberrans (Oudemans),A. aff.finlandicus (Oudemans),A. stipulatus Athias-Henriot,Phytoseiulus persimilis Athias-Henriot, andPhytoseius (Phytoseius) finitimus Ribaga.     

Phylogenetic Analysis of Sugarcane Rusts Based on Sequences of ITS, 5.8 S rDNA and D1/D2 Regions of LSU rDNA

Phylogenetic analysis of sugarcane rusts based on sequences of ITS and the 5.8 S rDNA revealed two highly divergent ITS groups among isolates of Puccinia sp. sensu Muta, 1987 and P. kuehnii specimens. Although there is sufficient divergence (exceeding normal intraspecific variation) between the ITS regions of the two groups to support separation into different species, unusually high homology of the ITS group I sequences with those of members of Cronartium and identical sequences of the D1/D2 regions of the LSU rDNA for all the isolates of “Puccinia sp.” and P. kuehnii that otherwise exhibited different ITS sequences, suggest that the two highly divergent sequences may have resulted from abnormal genetic events leading to non-orthologous, intraspeciflc polymorphisms. The other sugarcane rust, P. melanocephala and the grass rusts, P. miscanthi and P. rufipes, were separated from “Puccinia sp.” and P. kuehnii and from each other in D1/D2 region analyses, indicating that D1/D2 region sequences may more correctly reflect phylogenetic relationships in these rusts than do the ITS regions. Further studies to examine differences in patho-genicity or finer morphological features within P. kuehnii that may be correlated with the high divergence in ITS sequences and experiments to determine if these two sequence types represent intraspeciflc polymorphism are necessary. Received 11 October 2000/ Accepted in revised form 24 November 2000     

Identification and pathogenicity of Cryphonectriaceae species associated with chestnut canker in China

Cryphonectriaceae species cause serious canker diseases on chestnut, oak and eucalypt trees. Recently, canker symptoms with typical orange fruiting bodies were observed on Chinese chestnut and oak trees in Hebei, Hubei, Shaanxi and Shandong Provinces in China. In the present study, isolates of these fungi were identified based on phylogenetic and morphological evidence, and their pathogenicities were tested on detached chestnut (Castanea mollissima) branches. DNA sequence comparisons of the internal transcribed spacer (ITS) regions and two regions of β-tubulin (TUB1/TUB2) indicate that these isolates represent five species in the Cryphonectriaceae, viz. Cryphonectria japonica, Cryphonectria parasitica, Aurantiosacculus castaneae sp. nov., Cryphonectria neoparasitica sp. nov. and Endothia chinensis sp. nov. The sexual morph of Aurantiosacculus is discovered for the first time and can be distinguished from the other genera in Cryphonectriaceae by dark brown ascospores and tubiform appendages at both ends. Cryphonectria neoparasitica sp. nov. is different from the other Cryphonectria species by its aseptate ascospores. Endothia chinensis sp. nov. is the sole species in Endothia infecting the host genus Castanea. Additionally, it is much smaller than E. gyrosa and narrower than E. singularis in ascospores. The inoculation results showed that these five Cryphonectriaceae fungi isolated from chestnut or oak are all pathogenic to tested chestnut branches. Cryphonectria parasitica appears to be the most aggressive fungus, followed by C. neoparasitica sp. nov., C. japonica, E. chinensis sp. nov. and A. castaneae sp. nov.     

Molecular data from up to 130-year-old herbarium specimens do not support the presence of cherry powdery mildew in Australia

A strain of Podosphaera clandestina has been highlighted as a priority pest threat to the Australian cherry industry. Australia currently has no records of powdery mildew on cherry (Prunus avium). P. clandestina is reported to cause disease on a range of Rosaceae genera including Crataegus and Prunus; in Australia, P. clandestina has only been recorded on Crataegus. A recent species revision identified Podosphaera cerasi on P. avium as a separate species from P. clandestina. Therefore, a revision of which powdery mildew species is present in Australia on Crataegus is required to inform Australian plant biosecurity. Reference collection specimens from the Victorian Plant Pathology Herbarium (VPRI) recorded as Podosphaera spp. collected between 1889 to 2008 on cherry and three other host plant genera from Australia and overseas were sampled for DNA extraction and next-generation sequencing (NGS). Sequence data from preserved specimens were successfully mapped to internal transcribed spacer (ITS) sequences of P. clandestina in the strict sense, P. cerasi, and Podosphaera prunicola, and chloroplast matK sequences were used to identify plant hosts. Australian specimens on Crataegus hosts were P. clandestina in the strict sense and specimens on Prunus from the USA were identified as P. cerasi and P. prunicola. The outcome of this study confirmed the powdery mildew on Australian Crataegus specimens to be P. clandestina and none of the cherry powdery mildews (Podosphaera pruni-avium, P. cerasi, or P. prunicola) are present on Australian specimens in the VPRI collection, which suggests they are not present in Australia.     

Morphological and molecular characterisation of Pratylenchus oleae n. sp. (Nematoda: Pratylenchidae) parasitizing wild and cultivated olives in Spain and Tunisia

A new mono-sexual root-lesion nematode species, Pratylenchus oleae n. sp., parasitizing roots of olive plants cv. Koroneiki in commercial fields at Ouled Chamekh (central Tunisia), and wild and cultivated olive (cv. Picual) plants in Agua Amarga (southern Spain) is described. The new species is characterised by the female having a lip region slightly offset and bearing three annuli, stylet 16.5 (14.5-17.0) μm long, with prominent rounded knobs, pharyngeal overlapping rather long (22–36) μm, lateral fields areolated and with four incisures and diagonal lines in middle band, spermatheca rounded but non-functional, tail short, conoid-rounded to subcylindrical, usually annulated terminus, males unknown, and a specific D2-D3, ITS1, 18S-rRNA, hsp90 and COI sequences. Morphologically this species is related to P. cruciferus, P. delattrei, and P. kumamotoensis. The results of the phylogenetic analysis based on sequences of the D2-D3 expansion regions of 28S, partial 18S and ITS rRNA genes confirmed the close relationship of P. oleae n. sp. with P. dunensis, P. penetrans, P. pinguicaudatus, from which was clearly separated. A PCR-based diagnostic assay was also developed for identification of P. oleae n. sp. using the species-specific primers Poleae_fw1_4 and Poleae_rv1 that amplify a 547-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA, which clearly separate from other root-lesion nematodes damaging olive such as P. penetrans and P. vulnus.     

Characterisation and pathogenicity of Pestalotiopsis uvicola and Pestalotiopsis clavispora causing grey leaf spot of mango (Mangifera indica L.) in Italy

During 2009 and 2010, twenty-one isolates of Pestalotiopsis spp. Associated with grey patches on the leaves, twigs, and panicles of mango were collected in six orchards located in Sicily (Italy). Morphological characteristics of colony (colour and mycelium appearance), and conidia (size, shape, septation, length and the number of apical and the basal appendages) as well as phylogenetic analysis of the Internal Transcribed Spacer (ITS) region (ITS1, 5.8S gene, and ITS2) of six representative isolates revealed the occurrence of P. uvicola and P. clavispora. The representative isolates of both species were pathogenic to the artificially inoculated detached mango leaves cv. Kensington Pride and showed significant variation in lesion size. This is the first report worldwide of P. uvicola and P. clavispora causing grey leaf spot of mango.     

Prevalence of Phytophthora species in macadamia orchards in Australia and their ability to cause stem canker

In Australia, Phytophthora cinnamomi is the only species reported as the causal agent of stem canker and root rot in macadamia. In other countries, five Phytophthora species have been reported to cause diseases in macadamia, which led us to question if more than one Phytophthora species is responsible for poor tree health in macadamia orchards in Australia. To investigate this, samples were collected from the rhizosphere, stem, and root tissues of trees with and without symptoms, nurseries, and water sources from 70 commercial macadamia orchards in Australia. Phytophthora isolates were identified based on morphological characteristics and DNA sequencing. P. cinnamomi was the most predominant and widely distributed species, and was obtained from the different types of samples including symptomless root tissues. In addition to P. cinnamomi, only P. multivora was isolated from diseased tissue (stem canker) samples. Six other Phytophthora species were obtained from the rhizosphere samples: P. pseudocryptogea, P. citrophthora, P. nicotianae, P. gondwanense, P. sojae, and a new Phytophthora taxon. Only P. cinnamomi was obtained from macadamia nursery samples, while five Phytophthora species were obtained from water sources. Of the heterothallic Phytophthora species, mating type A2 isolates were dominant in P. cinnamomi isolates, whereas only mating type A1 isolates were obtained for P. nicotianae, P. pseudocryptogea, and P. citrophthora. Pathogenicity assays revealed that P. cinnamomi and P. multivora caused significantly larger stem and leaf lesions than P. citrophthora, P. nicotianae, and P. pseudocryptogea. Phytophthora sp. and P. sojae were nonpathogenic towards leaves and stems.     

Colletotrichum species associated with chili anthracnose in Australia

Phylogenetic relationships were determined for 45 Colletotrichum isolates causing anthracnose disease of chili in Queensland, Australia. Initial screening based on morphology, ITS and TUB2 genes resulted in a subset of 21 isolates being chosen for further taxonomic study. Isolates in the C. acutatum complex were analysed using partial sequences of six gene regions (ITS, GAPDH, ACT, CHS‐1, TUB2 and HIS3), and in the C. gloeosporioides complex were analysed using four gene regions (ITS, TUB2, ApMat and GS). Phylogenetic analysis delineated four Colletotrichum species including C. siamense, C. simmondsii, C. queenslandicum, C. truncatum and a new Colletotrichum species, described here as C. cairnsense sp. nov. This is the first reported association of C. queenslandicum, C. simmondsii and C. siamense with chili anthracnose in Australia; these species were previously associated with anthracnose on papaya and avocado. Furthermore, the dominant species causing anthracnose of chili in Southeast Asia, C. scovillei, was not detected in Australia. Inoculations on chili fruit confirmed the pathogenicity of C. cairnsense and the other four species in the development of chili anthracnose in Australia.     

Atripes paspali gen. et sp. nov. (Magnaporthaceae) causing take-all disease on Paspalum guenoarum in Brazil

In Brazil, Paspalum species are commonly used in sports lawns, landscape projects, and as forage for livestock. Paspalum guenoarum plants showing symptoms of take-all disease were observed in the state of Rio Grande do Sul, Brazil. The fungus Gaeumannomyces graminis is the only species reported associated with this disease on Paspalum. In recent years, new species of Gaeumannomyces have been proposed based on molecular studies, which demonstrated the existence of a species complex. Take-all affects rice and wheat, but the aetiology of this disease on P. guenoarum is still unknown; this work aimed to elucidate the aetiology of the take-all on P. guenoarum in Brazil and evaluate possible alternative hosts of agricultural importance. Based on combined phylogenetic analyses of ITS, LSU, TEF-1α, and RPB1 sequences, the fungal pathogen was identified as Atripes paspali gen. et sp. nov., which is proposed as a new genus in the Magnaporthaceae family. A representative isolate of A. paspali was inoculated on healthy P. guenoarum plants and reproduced the same symptoms of take-all observed in the field. Furthermore, this fungus is also able to cause take-all on wheat plants; temperature directly affected the incidence and development of the disease in wheat. Take-all on P. guenoarum is caused by A. paspali.     

Pewenomyces kutranfy gen. nov. et sp. nov. causal agent of an important canker disease on Araucaria araucana in Chile

Araucaria araucana, (commonly referred to as araucaria, pewen, or monkey puzzle tree) is an ancient conifer endemic to the Chilean and Argentinian mountain ranges where it has a sacred relevance to indigenous communities. During 2015, a serious disease was noticed on trees of all ages in most of the natural distribution of this iconic tree. Four areas were surveyed, and the most important symptoms of the disease were cankers on branches and stems resulting in copious resin exudation. Trees were monitored for a period of two years and isolations were made from the cankers. Field observations showed that the disease typically begins on the leaves or at the leaf bases and progresses downwards to initiate cankers that can girdle branches or stems within a two-year period. Black ascomata, resembling those of Caliciopsis species previously described from A. araucana, were consistently found developing in the cankers from which isolations were made. Phylogenetic analyses of the ITS, nucSSU, and nucLSU gene regions showed that the fungus resides in the Coryneliaceae but is distinct from other genera in that family. The morphological characteristics and phylogenetic position of the fungus show that it represents a new genus and species, described here as Pewenomyces kutranfy gen. nov. et sp. nov. Pathogenicity trials on trees under field conditions confirmed that this newly described fungus is able to cause cankers on A. araucana similar to those found under natural conditions.     

Molecular analysis of Phytophthora diversity in nursery‐grown ornamental and fruit plants

The genetic diversity of Phytophthora spp. was investigated in potted ornamental and fruit tree species. A metabarcoding approach was used, based on a semi‐nested PCR with Phytophthora genus‐specific primers targeting the ITS1 region of the rDNA. More than 50 ITS1 sequence types representing at least 15 distinct Phytophthora taxa were detected. Nine had ITS sequences that grouped them in defined taxonomic groups (P. nicotianae, P. citrophthora, P. meadii, P. taxon Pgchlamydo, P. cinnamomi, P. parvispora, P. cambivora, P. niederhauserii and P. lateralis) whereas three phylotypes were associated to two or more taxa (P. citricola taxon E or III; P. pseudosyringae, P. ilicis or P. nemorosa; and P. cryptogea, P. erythroseptica, P. himalayensis or P. sp. ‘kelmania’) that can be challenging to resolve with ITS1 sequences alone. Three additional phylotypes were considered as representatives of novel Phytophthora taxa and defined as P. meadii‐like, P. cinnamomi‐like and P. niederhauserii‐like. Furthermore, the analyses highlighted a very complex assemblage of Phytophthora taxa in ornamental nurseries within a limited geographic area and provided some indications of structure amongst populations of P. nicotianae (the most prevalent taxon) and other taxa. Data revealed new host–pathogen combinations, evidence of new species previously unreported in Italy (P. lateralis) or Europe (P. meadii) and phylotypes representative of species that remain to be taxonomically defined. Furthermore, the results reinforced the primary role of plant nurseries in favouring the introduction, dissemination and evolution of Phytophthora species.     

Diversity of Botryosphaeriaceae species associated with conifers in Portugal

Botryosphaeriaceous fungi were isolated from conifers showing disease symptoms such as diebacks, blights, and cankers. The isolates were grouped based on morphology and ERIC-PCR fingerprinting patterns and representatives of each group were identified by sequencing of the internal transcribed spacer (ITS) region. Nine species from four different genera in Botryosphaeriaceae were identified within the isolates. Many new fungus-host associations were established and several species of Botryosphaeriaceae are reported from conifers for the first time. Most of these species also represent new reports from Portugal. The genus Neofusicoccum that was thought to be mainly restricted to angiosperms was the most frequent within the collection of isolates, followed by Diplodia. Dothiorella and Botryosphaeria represented a minor fraction of the isolates. Interestingly, the most common species was N. luteum, which had never been reported from coniferous hosts. Our results indicate that Neofusicoccum species may be more important as pathogens of conifers than it was previously recognised.     

Death of endemic Virgilia oroboides trees in South Africa caused by Diaporthe virgiliae sp. nov.

Numerous dead and dying individuals of the Western Cape endemic tree Virgilia oroboides (Fabaceae) were recently observed within a South African national botanical garden. Root‐rot fungi and fungi symbiotic with bark beetles (Curculionidae; Scolytinae) from diseased trees were assessed for their respective roles in V. oroboides mortality. Disease progression was also monitored over 1 year. Fungi were isolated from surface sterilized bark and root samples from diseased trees and provisionally identified using data from the internal transcribed spacer regions (ITS1, ITS2), including the 5·8S rRNA gene (ITS). Pathogenicity of selected fungi towards V. oroboides was tested under field conditions. The pathogenicity of various bark beetle‐associated Geosmithia (Hypocreales: Hypocreomycetidae) spp. from V. oroboides were similarly assessed. The only fungus consistently isolated from lesions on the roots and bark of declining V. oroboides, and never from healthy individuals, represented an undescribed Diaporthe (Diaporthales, Diaporthaceae) species that was characterized using molecular (using data from the ITS marker and part of the β‐tubulin gene, TUB), cultural and morphological characters. It is an aggressive pathogen of V. oroboides, newly described here as Diaporthe virgiliae sp. nov. Trees of all ages are susceptible to this pathogen with subsequent bark beetle attack of mature trees only. All Geosmithia spp. from beetles and/or infected trees were nonpathogenic towards V. oroboides. Diaporthe virgiliae caused a severe decline in the health of the monitored V. oroboides population over a period of only 1 year and should be considered as a significant threat to these trees.     

Occurrence of soilborne Phytophthora species in declining broadleaf forests in Hungary

Invasive Phytophthora species are responsible for severe tree diseases in many forest ecosystems in Europe. In Hungary, the symptoms were first noted when P. alni infection led to a serious decline and mortality of alder stands in the late 1990s. Between 2001 and 2009, over 300 soilborne Phytophthora isolates were collected from declining broadleaf forests in Hungary, and 10 Phytophthora species identified based on morphological traits and the molecular characteristics of the internally transcribed spacer (ITS) regions of the nuclear ribosomal DNA. The most diverse species spectrum, found in diseased alder stands, included P. gonapodyides, P. gregata, P. inundata, P. lacustris, P. megasperma, P. plurivora, one informally designated taxon: P. taxon hungarica, and one unnamed species P. sp.1. P. cactorum and P. plurivora isolates were prevalent in the soil of a declining eastern black walnut forest, and three species, P. gonapodyides, P. multivora and P. plurivora were recovered from a declining oak stand. More than one ITS-based genotype was identified for four species, including six genotypes for P. gonapodyides, and two each for P. cactorum, P. plurivora and P. inundata. The high genetic diversity of the P. gonapodyides isolates may indicate that the species is indigenous to the region. In contrast, the frequently recovered, widely distributed P. lacustris with a single ITS genotype may represent a recent colonizer. The P. multivora isolates are, to date, the first reported from a European native forest.     

Phylogeny, diversity, and species delimitation in some species of the Xiphinema americanum-group complex (Nematoda: Longidoridae), as inferred from nuclear and mitochondrial DNA sequences and morphology

During nematode surveys in southern Spain and Italy 14 populations of Xiphinema species tentatively identified as Xiphinema americanum-group were detected. Morphological and morphometrical studies identified three new species and six known Xiphinema americanum-group species, viz.: Xiphinema parabrevicolle n. sp., Xiphinema parapachydermum n. sp., Xiphinema paratenuicutis n. sp., Xiphinema duriense, Xiphinema incertum, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema rivesi, and Xiphinema santos. The Xiphinema americanum-group is the most difficult Xiphinema species group for diagnosis since the morphology is very conservative and morphometric characters often overlap. This group includes vectors of several important plant pathogenic viruses that cause significant damage to a wide range of agricultural crops. Molecular characterisation of these species using D2-D3 expansion regions of 28S rRNA, 18S rRNA, ITS1-rRNA and the protein-coding mitochondrial gene, cytochrome oxidase c subunit 1 was carried out and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships among these species and with other Xiphinema americanum-group species.