Pathogenicity of Phytophthora alni complex and P. plurivora in Alnus glutinosa seedlings

The ecological importance of riparian forests is well known. However, these forest habitats have been disturbed by human activities over the past century as a result of the introduction of flow regulations. Mortality of the riparian alder population caused by Phytophthora has become an important issue in Europe in the past two decades. The main objective of this study was to examine the pathogenicity of the Phytophthora alni complex (P. xalni, P. uniformis and P. xmultiformis) and P. plurivora in Alnus glutinosa seedlings. Phytophthora alni complex has traditionally been identified as the main causal agent of alder decline; however, in this study, P. plurivora was found to cause as much damage in inoculated seedlings as P. alni complex. In fact, Spanish isolates of P. xalni caused mortality rates of ca. 30%, whereas P. plurivora killed ca. 50% of seedlings. Likewise, there were no differences between P. xalni ‐ and P. plurivora‐inoculated seedlings in either the length of lesion or biomass allocation. The pathogenicity of the species within P. alni complex did not differ significantly. The findings confirm that P. plurivora, which is one of the most common species of Phytophthora found in European nurseries, is highly pathogenic to alder seedlings. Urgent implementation of measures to prevent the risk of spread of the pathogen from nurseries to natural ecosystems is required. Further studies are required to clarify the role that P. plurivora is playing in alder decline both individually and in combination with P. alni complex.     

Pathogenicity of Malaysian Phytophthora palmivora on cocoa,durian, rubber and oil palm determines the threat of bud rot disease

The epidemic of bud rot disease affecting oil palm in Colombia is primarily caused by Phytophthora palmivora. The pathogen has a cosmopolitan presence that includes Southeast Asia, but to date, bud rot has not been reported in this region. This study provides an overview of the potential risk of Malaysian P. palmivora isolates cross‐infecting other host species, including cocoa, durian, rubber and Malaysian oil palm planting materials (Dura × Pisifera, D × P). On cocoa pods, the durian isolate PP7 caused dark brown necrotic lesions. Detached leaf bioassays showed that P. palmivora isolates PP3 and PP7 infected different hosts, except rubber foliage without wounding. Inoculation tests on cocoa, durian and rubber seedlings caused brown necrotic lesions when stems were wounded, with 10% mortality in cocoa and durian at 17 days post‐inoculation (dpi). However, no further infection was observed, and lesions closed within 14–28 dpi on the non‐wounded seedlings. Pathogenicity tests of oil palm seedlings inoculated with isolates PP3 and PP7 indicated that Malaysian P. palmivora isolates were not pathogenic to oil palms based on localized infection observed only through wounding. Overall, the work demonstrated that Malaysian P. palmivora isolates were able to cross‐infect multiple hosts but did not show severe infections on oil palms.     

Pathogenicity to alder of Phytophthora species from riparian ecosystems in western Oregon

Described as one of the most destructive pathogens of agricultural crops and forest trees, Phytophthora is a genus of microorganisms containing over 100 known species. Phytophthora alni has caused collar and root disease in alders throughout Europe, and a subspecies has recently been isolated in North America. Reports of canopy dieback in red alder, Alnus rubra, prompted a survey of their overall health and to determine whether P. alni was present in western Oregon riparian ecosystems. Over 1100 Phytophthora isolates were recovered, representing 20 species and 2 taxa. Phytophthora‐type cankers were observed in many trees, and their incidence was positively correlated with canopy dieback. High levels of mortality for red alder were not observed, which suggests these Phytophthora species are not aggressive pathogens. To test this hypothesis, three stem wound inoculations and one root dip were conducted on red alder seedlings using 13 Phytophthora species recovered from the riparian survey. Ten of the 13 Phytophthora species produced significant lesions in at least one pathogenicity test. Phytophthora siskiyouensis produced the largest lesions on red alder from the two stem wound inoculation tests conducted under summer conditions, while P. taxon Pgchlamydo caused the largest lesions during the winter stem wound inoculation test. Phytophthora gonapodyides, P. taxon Pgchlamydo and P. siskiyouensis have previously been found associated with necrotic alder roots and bole cankers in the field, and with the pathogenicity results reported here, we have established these species as causes of Phytophthora root disease and Phytophthora bole canker of alder in Oregon. While none of the Phytophthora species were especially aggressive towards red alder in the pathogenicity tests, they did cause localized disease symptoms. By weakening the root systems or boles of alders, the Phytophthoras could be leaving alders more susceptible to other insects and pathogens.     

Current status of the Dutch elm disease pathogen populations affecting Ulmus minor in Greece

One hundred and eight Ophiostoma ulmi sensu lato isolates were collected from field elm trees with symptoms in 14 Prefectures of Greece. The purpose of this study was to assign Greek isolates to species and afterwards subspecies of the DED fungi and to analyse the genetic variability within the Greek populations of these pathogens. Isolates were compared with six reference strains belonging to O. ulmi and the two subspecies of O. novo‐ulmi. The structure of the population has been analysed by means of morpho‐physiological features (growth rates, colony morphology and fertility responses) and by DNA sequencing and PCR‐RFLP amplification of the cerato‐ulmin (cu) and the colony type (col1) gene regions. According to fertility tests, both subspecies of O. novo‐ulmi were detected in Greece, but none of the isolates collected was identified as O. ulmi. O. novo‐ulmi ssp. novo‐ulmi occurred more frequently than ssp. americana (73 and 35 isolates, respectively) and their ranges overlapped. All isolates that behaved as ssp. novo‐ulmi in the fertility tests had the cu, as well as the col1 profile of ssp. novo‐ulmi. Surprisingly, all isolates that behaved as O. novo‐ulmi ssp. americana in the fertility test had the cu, as well as the col1 (with one exception) profile of O. novo‐ulmi ssp. novo‐ulmi. A possible explanation for this inconsistency could be the occurrence of hybridization between the two subspecies in Greece.     

Temporal metabolic profiling of the Quercus suber–Phytophthora cinnamomi system by middle‐infrared spectroscopy

The oomycete Phytophthora cinnamomi is an aggressive plant pathogen, detrimental to many ecosystems including cork oak (Quercus suber) stands, and can inflict great losses in one of the greatest ‘hotspots’ for biodiversity in the world. Here, we applied Fourier transform‐infrared (FT‐IR) spectroscopy combined with chemometrics to disclose the metabolic patterns of cork oak roots and P. cinnamomi mycelium during the early hours of the interaction. As early as 2 h post‐inoculation (hpi), cork oak roots showed altered metabolic patterns with significant variations for regions associated with carbohydrate, glycoconjugate and lipid groups when compared to mock‐inoculated plants. These variations were further extended at 8 hpi. Surprisingly, at 16 hpi, the metabolic changes in inoculated and mock‐inoculated plants were similar, and at 24 hpi, the metabolic patterns of the regions mentioned above were inverted when compared to samples collected at 8 hpi. Principal component analysis of the FT‐IR spectra confirmed that the metabolic patterns of inoculated cork oak roots could be readily distinguished from those of mock‐inoculated plants at 2, 8 and 24 hpi, but not at 16 hpi. FT‐IR spectral analysis from mycelium of P. cinnamomi exposed to cork oak root exudates revealed contrasting variations for regions associated with protein groups at 16 and 24 h post‐exposure (hpe), whereas carbohydrate and glycoconjugate groups varied mainly at 24 hpe. Our results revealed early alterations in the metabolic patterns of the host plant when interacting with the biotrophic pathogen. In addition, the FT‐IR technique can be successfully applied to discriminate infected cork oak plants from mock‐inoculated plants, although these differences were dynamic with time. To a lesser extent, the metabolic patterns of P. cinnamomi were also altered when exposed to cork oak root exudates.     

Epitypification,characterization and phylogenetic positioning of Pseudobeltrania cedrelae,the causal agent of pseudobeltrania spot on Cedrela fissilis

The fungus Pseudobeltrania cedrelae, the type species of the genus, is the causal agent of an important leaf spot in seedlings and adult plant of cedar (Cedrela fissilis). Due to the contradictory phylogenetic position of the genus Pseudobeltrania, epitypification of P. cedrelae was carried out based on a culture obtained from the same locality and host of the original type. Samples were collected, and 10 isolates of P. cedrelae associated with lesions on cedar leaflets were obtained. For morphological characterization, conidia, conidiophores, conidiogeneous cells, conidiogeneous loci and basal cells were taken both from the fungus obtained from leaf lesions and from that obtained in slide culture. Mycelial growth rates and sporulation were evaluated in six different culture media. For molecular phylogeny, maximum parsimony analyses were performed from the ITS and 28S sequences of the isolates. Both the morphological characteristics of the fungal structures obtained from symptomatic leaves and the slide culture technique presented variations. In foliar lesions, isolates presented the same morphological characteristics as the type material. Mycelial growth rate and sporulation of P. cedrelae were greatest on malt extract agar and V8 juice agar. Pseudobeltrania cedrelae was pathogenic when inoculated into healthy cedar plants. According to the phylogenetic tree, isolates grouped in the same clade, but in a distinct clade of Pseudobeltrania ocoteae. The results suggested that P. ocoteae belongs to the genus Hemibeltrania. This paper presents new information on P. cedrelae that contributes to clarifying the phylogeny of the Beltraniaceae.     

Leaf necrosis and sucker and twig dieback of Alnus glutinosa incited by Pseudomonas syringae pv. syringae.

In central Italy, Pseudomonas syringae pv. syringae was isolated from Alnus glutinosa that showed, in spring, leaf necrosis and also sucker and twig dieback. The trees were located near a lake and a stream. Biochemical tests were consistent with those characterizing the pathovar. Pathogenicity tests yielded necrotic lesions on A. glutinosa and Syringa vulgaris leaves and also on lemon and orange fruits. In addition, whole-cell protein profiles of the isolates and P. s. pv. syringae reference strains were identical. Some isolates showed positive ice-nucleation activity. This is the first record of this pathogen on black alder.     

Pinus taeda L. response to differential inoculum density of Leptographium terebrantis colonized toothpicks

Bark beetle‐vectored ophiostomatoid fungi, Leptographium terebrantis, is inoculated on the roots and lower stems of stressed Pinus species during the feeding activity of bark beetle. To determine the exact host response following inoculation, it is critical to challenge the host with a realistic amount of fungal inoculum. Thus, we designed a series of stepwise experiments using L. terebrantis colonized toothpicks which focused on the inoculum transfer from the toothpicks to excised Pinus taeda stem segments and living saplings, respectively, at different inoculum densities. The toothpicks served as a substrate for fungal growth and sporulation and the inoculation showed their utility in eliciting host's response to the pathogen. The inoculated fungus caused blue‐stain and sapwood occlusions in P. taeda stems and saplings, respectively. The volume of occluded, visually damaged sapwood increased by 1.96 cm³ per radial inoculation point on average. Fungal colonized toothpicks can be used as a suitable alternative to agar discs for studying bark beetles vectored fungi and their host interactions.     

High‐resolution melting analysis allows rapid and accurate differentiation of the three Melampsoridium species occurring on Alnus

Alder (Alnus) leaves can be infected by three species of Melampsoridium rust: M. alni, M. betulinum and M. hiratsukanum. The latter is an invasive species in Europe that can be differentiated from the other two on the basis of morphological and molecular characters. This study presents a rapid, one‐step, closed‐tube and inexpensive method to differentiate species of Melampsoridium using two primer sets and high‐resolution melting (HRM) analysis of alder leaf material.     

Fruiting and sporulation of Thekopsora and Chrysomyxa cone rusts in Picea cones and Prunus leaves

Natural fruiting and sporulation of cone rusts were investigated in cones of Picea spp. and leaves of Prunus spp. in a botanical garden in northern Finland in 2007–2012. Thekopsora areolata was the most frequent cone rust in Picea abies cones, where it colonizes the host tissues and hinders normal seed development. Aecia of T. areolata were also common in cones of Picea engelmannii and occasionally in cones of P. glauca. Aecia of T. areolata sporulated in cones that were at least one year old. Chrysomyxa pirolata, another pathogenic cone rust, fruited and sporulated annually but infrequently in current‐year cones of P. abies. The spruce needle rust, Chrysomyxa ledi, fruited and sporulated commonly in current‐year cone scales of P. abies, P. omorika and P. glauca, while P. rubens, P. mariana and P. pungens appeared to be resistant during the study period. Chrysomyxa ledi did not affect seed development in infected cones. Uredinia of T. areolata frequently occurred on leaves of 41 Finnish and Russian cultivars, varieties or subspecies of Prunus padus L. ssp. badus and ssp. borealis and Pr. virginiana both in the botanical garden and in the field, while 13 exotic Prunus spp. lacked rust fruitbodies. All the Pr. padus cultivars were highly susceptible to T. areolata, thus, spreading the rust efficiently to surroundings. This is the first report of aecia of T. areolata in cones of P. engelmannii and P. glauca, and those of C. ledi in cones of P. omorika and P. glauca. Molecular identification confirmed the presence of T. areolata and C. pirolata on all hosts, and all samples of C. ledi belonged to the C. ledi‐rhododendri complex.     

Foliar susceptibility of eastern oak species to Phytophthora infection

Seven different Phytophthora species were used to test the foliar susceptibility of the common eastern US oak species and understory plants to Phytophthora infection. The Phytophthora species employed were Phytophthora cambivora, Phytophthora cinnamomi, Phytophthora citricola, Phytophthora europaea, Phytophthora quercetorum, Phytophthora quercina‐like and Phytophthora sp1. Inoculation of detached‐leaves with agar plugs containing mycelia of Phytophthora provided an estimate of their relative susceptibility. Lesions were always greater when foliage was wounded and young. On deciduous plants, lesion sizes were considerably reduced with the increasing foliar age, although with evergreen plants lesion sizes remained similar regardless of foliar age when more aggressive isolates were tested. Infections seldom resulted when foliage was not wounded. With young and mature foliage, P. citricola usually produced the largest lesions. Young foliage of Quercus rubra was the most susceptible to infection followed by Castanea dentata for both wounded and non‐wounded inoculations. Mature foliage of Hamamelis virginiana, Kalmia latifolia and Quercus alba were the most susceptible to wound and non‐wound inoculations.     

Phytophthora ×alni and Phytophthora lacustris associated with common alder decline in Central Portugal

Since the early 1990s, an emerging disease induced by the highly aggressive oomycete Phytophthora ×alni has caused widespread alder decline across Europe. In parallel, P. lacustris, a recently described species associated with riparian habitats, has been subject of increasing interest. A field survey conducted in 2014 showed high mortality rates in alder stands located in the riparian gallery along two rivers in Central Portugal. The pathogens isolated from necrotic alder stem base during this study were identified as P. ×alni and P. lacustris. This paper is the first to report the occurrence of P. lacustris in Portugal and presents the first finding of P. ×alni affecting mature trees in natural ecosystems located in Central Portugal.     

A new leaf disease on Populus euphratica in south‐western Iran

During the winter of 2012, a leaf spot disease was observed on Euphrates poplar (Populus euphratica) in the forest areas of Khuzestan province, south‐western Iran, causing significant damage in the Karun's riverside forests. Symptoms consisted of necrotic dark brown, circular to oval, 5‐ to 10‐mm spots on both surfaces of the leaves. A fungus having distinct dictyospores similar to those produced by Alternaria spp. was observed. The morphological characteristics, as well as the phylogenetic analysis of the internal transcribed spacer (ITS1‐5,8S‐ITS2) region, confirmed the identity of the strains belonging to the species Alternaria alternata. Pathogenicity tests were conducted on the alive leaves of P. euphratica on the young branches, as well as on the detached leaves in Petri dishes, through inoculation with spore suspension. Target spot symptoms similar to those observed in naturally infected leaves were developed on the inoculated leaves seven to 10 days after inoculation in both the inoculation procedures. A. alternata was consistently re‐isolated from the spots. Interestingly, similar symptoms were observed 7 days after detached leaf treatment with droplets of 15‐day‐old fungal culture filtrate, suggesting the production of pathotoxic compounds by the fungus. To our knowledge, this is the first report of A. alternata causing leaf spot on Euphrates poplar in Iran.     

Fusarium species in declining wild apple forests on the northern slope of the Tian Shan Mountains in north‐western China

Wild apple forests in the Tian Shan Mountains in north‐western China have been adversely affected by an unknown disease in recent years. Symptoms attributed to this disease that affects wild apple trees include xylem browning and dieback which are suggestive of infection by Fusarium species. Therefore, the research team conducted the first survey for Fusarium in the afflicted wild apple forests. Twig samples with symptoms of xylem browning and dieback were collected in the Xinyuan, Gongliu, Yining and Huocheng Counties of Xinjiang Uighur Autonomous Region in China. Based on phylogenetic analyses and morphological observation, sixty strains of Fusarium accounted for 48% of the total number of fungi isolated from samples were subsequently classified into six species including twenty‐four F. avenaceum, seventeen F. solani, ten F. tricinctum, five F. proliferatum, two F. sporotrichioides and two unfamiliar Fusarium sp. 1. The five previously known species of Fusarium were then tested for pathogenicity to leaves and twigs in vitro. The results indicated that all of the species, except for F. tricinctum, can cause obvious lesions on the leaves of host plants and on the twigs of Fuji and wild apple. This is the first report of Fusarium species pathogenicity in Xinjiang wild apple forests, confirming a new host for these pathogens in this study.     

A twig blight of understorey European beech (Fagus sylvatica) caused by soilborne Phytophthora spp.

During and after prolonged periods of rainfall in late spring, blighted young twigs of European beech (Fagus sylvatica) were frequently observed in several beech stands in south‐western and southern Germany. Long and short shoots of young understorey trees or lower branches up to 1.5 m above the soil level were affected. Symptoms also occurred regularly on twigs in heights up to 2 m and more above the ground. Necroses usually expanded within the current year’s tissue and often also reached into the previous year’s wood. Ponding rain water in the stands or along forest roads or open soil seemed to promote the disease. Of a total of 54 symptomatic twigs collected in four stands, 37 revealed Phytophthora isolates, of which 33 were P. plurivora and four were P. cambivora. Both species caused extensive lesions on beech twigs in laboratory pathogenicity tests. Patterns of the disease indicated that these pathogens, generally considered soilborne species, in most cases are transmitted from the soil to above‐ground parts of the trees via rain splash. In larger heights, however, other vectors such as snails might be responsible for transmission. Although Phytophthora spp. are well known as causal agents of seedling blight as well as root and cambium rot and aerial bleeding cankers of mature beech, to our knowledge this is the first report of a twig blight in beech associated with soilborne Phytophthora spp. In particular in periods of high precipitation, this disease might pose an additional threat to Central European beech forests, especially endangering the success of artificial and natural regeneration of beech in affected stands.     

Pathogenicity of Heterobasidion annosum (Fr.) Bref. sensu stricto on coniferous tree species in Turkey

Two‐year‐old seedlings of Pinus brutia, P. brutia var. eldarica, Pinus pinea and 3‐year‐old seedlings of Pinus radiata, Pinus sylvestris, Pinus nigra and Cedrus libani were inoculated on the lower stem with isolates of Heterobasidion annosum s.s. collected from the Black Sea and Mediterranean regions of Turkey. In total, 315 seedlings were inoculated in April 2014 and incubated in a growth chamber for 7 weeks at 18–20°C. All isolates were pathogenic on the seven different hosts and had the ability to grow in living sapwood. The isolates had a greater growth on C. libani, P. sylvestris and P. radiata seedlings compared to plants of the other species tested. The least affected species were P. brutia and P. nigra. The isolates originating from the Black Sea region caused longer lesions on the hosts. Overall mortality during 7 weeks of incubation was 4%.     

Susceptibility of Pedicularis spp. to Cronartium ribicola and C. flaccidum in Finland

Aeciospores of Cronartium ribicola and C. flaccidum were collected from several locations in Finland and used to inoculate Pedicularis spp. and some known and suspected alternate hosts in 2008–2009. In all trials, C. ribicola formed uredinia and telia on leaves of Ribes nigrum. No uredinia or telia of C. ribicola formed on older leaves of Pedicularis spp. but both uredinia and telia were found on young leaves of P. palustris ssp. palustris. Cronartium flaccidum produced uredinia and/or telia on leaves of P. palustris ssp. palustris, P. lapponica, Vincetoxicum hirundinaria and Melampyrum sylvaticum. Neither rust infected P. sceptrum‐carolinum, Vaccinium myrtillus, Calluna vulgaris or Ledum palustre. Similar to rusts in Asia and North America, the results showed that European C. ribicola exhibit more variable host reactions and wider alternate host ranges than earlier described. Pedicularis palustris may play a role in the spread of Cronartium in natural forests.     

Lesion development in stems of rough‐ and smooth‐barked Eucalyptus nitens following artificial inoculations with canker fungi

A study of lesion development in stems of Eucalyptus nitens following artificial inoculations with canker fungi was carried out on 16‐year‐old plantation trees. In a first trial cambium bark wounds on smooth‐ and rough‐barked trees were inoculated with the mycelium of nine species of canker fungi, including Endothia gyrosa. In a second trial spores or mycelium of E. gyrosa were applied directly onto undamaged or superficially wounded bark surfaces. Infection subsequent to artificial inoculation via wounding (whatever the wounding technique or type of inoculum) resulted in significantly larger external lesions (mean lesion area up to 35.6 cm² 20 months after inoculation) on smooth bark compared with those on rough bark (up to 19.0 cm²). Microscopic studies of infected rough and smooth bark suggest that, once smooth bark is compromised by wounding and artificial inoculation, the particular anatomical structure of smooth bark may offer less mechanical resistance to post‐penetration hyphal spread in comparison with rough bark. It is suggested that at a pre‐penetration stage under natural conditions spores of E. gyrosa more easily infect rough bark via cracks associated with this type of bark but not present in smooth bark.     

Alder rust,Melampsoridium hiratsukanum Ito,identified from Wales,UK and British Columbia,Canada

An epidemic rust, Melampsoridium hiratsukanum, originating from East Asia has spread to Europe over the last two decades. We report the first observation of this rust in the British Isles on leaves of Alnus incana and A. glutinosa. In addition to M. hiratsukanum, a native M. betulinum–like rust co‐occurred on the same leaves as a mixed infection. We also confirm that a rust on A. rubra in Western Canada is identical to M. hiratsukanum present in East Asia and Europe.     

Cultural characteristics and pathogenicity of Pestalotiopsis funerea on Cupressus arizonica

The fungal pathogen Pestalotiopsis funerea principally attacks conifer species, causing necrosis on infected tissues, and sometimes death of plants. The main objectives of this study were to determine optimal in vitro growth conditions for Spanish isolates of P. funerea and to evaluate the virulence of these isolates on Cupressus arizonica under field and laboratory conditions, with the goal of verifying possible correlations between these variables. Eight isolates of P. funerea derived from C. sempervirens, C. arizonica and Quercus pyrenaica were used in the assays. In the growth rate experiment, five culture media (PDA, MEA, WA, PCA and TAKAY medium) and six temperatures (5, 10, 15, 20, 25 and 30°C) were evaluated. In the pathogenicity tests, two different experiments were carried out: (i) laboratory inoculations used 30‐mm‐long twigs of C. arizonica; (ii) in field inoculations, twigs of C. arizonica were inoculated with mycelia through wounds. Four months after inoculation, length of the twigs affected by necrosis were measured. The results suggested that Spanish P. funerea isolates had optimum growth at 25°C on TAKAY medium. The fungus caused substantial necroses on C. arizonica twigs after inoculation in both excised twigs and on adult C. arizonica trees. A positive relationship between inoculations carried out under laboratory and field conditions was found. No clear relationships were found between cultural characteristics and pathogenicity for P. funerea.