Development of a quantitative real‐time PCR assay for the detection of Phytophthora austrocedrae,an emerging pathogen in Britain

A TaqMan real‐time PCR assay was developed for Phytophthora austrocedrae, an emerging pathogen causing severe damage to juniper in Britain. The primers amplified DNA of the target pathogen down to 1 pg of extracted DNA, in both the presence and absence of host DNA, but did not amplify any of the non‐target Phytophthora and fungal species tested. The assay provides a useful tool for screening juniper populations for the disease.     

Extent of colonization by Raffaelea quercivora of artificially inoculated living and gamma‐ray‐sterilized seedlings of two Japanese and three American oak species

Mass mortality of Fagacean tree species caused by Raffaelea quercivora has occurred widely in Japan. Because conidia or other propagules of the pathogen have not been found in infected trees, pathogen spread is assumed to occur primarily by hyphae. To clarify the relationship between hyphal growth of the pathogen within trees and their vessel arrangements, we examined two native Japanese oaks, Quercus crispula and Quercus glauca, and three exotic American oaks, Quercus coccinea, Quercus palustris and Quercus rubra. Quercus glauca is a radial‐porous species, whereas the other four species have a ring‐porous wood structure. Hyphal growth within inoculated potted living seedlings and in cut, sterilized stem segments of these species was examined microscopically after fungal inoculation. Water conductance in the seedlings was examined using transverse stem sections. The proportion of non‐conductive sapwood in Q. crispula, Q. coccinea and Q. palustris differed between inoculation and control treatment, being much higher in inoculated seedlings. The proportions were positively correlated with the extent of the hyphal growth. In sterilized stem segments, the extent of fungal colonization varied among the foreign ring‐porous species Q. coccinea, Q. palustris and Q. rubra. It is hypothesized that the extent of colonization by R. quercivora reflects the extent of non‐conductive sapwood irrespective of tree species, but is little affected by vessel arrangements.     

The northernmost plant pathogenic fungus,Rhytisma arcticum sp. nov.: Morphological and molecular characterization of a novel species from Ellesmere Island,Canada

A novel fungal pathogen, Rhytisma arcticum, is described, causing tar spot disease on Salix arctica in Ellesmere Island, Canada. Stromata, asci and ascospores—distinctly smaller than other Rhytisma species found on Salix—are its distinctive morphological features. rDNA internal transcribed spacer (ITS) and large-subunit (LSU) sequence analyses revealed a close relation of this species to R. polare previously reported on Spitsbergen Island. This study describes a novel plant pathogen species in one of the northernmost locations of the world.     

Detection and characterization of Ralstonia pseudosolanacearum infecting Eucalyptus sp. in Brazil

Ralstonia solanacearum sensu lato causes bacterial wilt in many agronomic crops and tree species economically important worldwide. It is a species complex that has been divided into phylotypes and sequevars, commonly related to geographic distribution. Knowledge of the phylotype composition and genetic variability in populations of this phytopathogenic bacterium is useful for implementing effective control measures. In a survey conducted in 2019, six bacterial strains were obtained from wilted Eucalyptus urophylla trees in plantations located in the municipality of Dom Eliseu, Pará state, Brazil. Multiplex PCR based on the internal transcribed spacer (ITS) indicated that the bacterial strains belonged to two different species, namely R. pseudosolanacearum (phylotype I) and R. solanacearum (phylotype II). In a phylogenetic analysis, the nucleotide sequence of the endoglucanase (egl) gene from eucalypt strains of phylotype I clustered together with sequevar 18 sequences from GenBank. Separation of the strains into two different species was confirmed by repetitive element palindromic PCR (rep‐PCR). Pathogenicity tests demonstrated that the R. solanacearum and R. pseudosolanacearum strains recovered from E. urophylla cause disease in both tomato and eucalypt plants. Until now, only R. solanacearum (Phylotype II) has been reported causing wilt symptoms on Eucalyptus spp. in Brazil. Therefore, the presence of R. pseudosolanacearum and a need for better understanding of its genetic and aggressiveness variability as well as possible differences between the two species should be considered in breeding programmes aimed at the deployment of host resistance.     

Colletotrichum siamense: A novel leaf pathogen of Sterculia nobilis Smith detected in China

Sterculia nobilis is an important tropical woody plant with high ornamental and economic value. At present, there are few reports of diseases on this plant. In August 2018, an unknown leaf spot disease was observed on S. nobilis in Nanning, China, affecting the inner leaves of the canopy and causing considerable defoliation. The pathogen was isolated, and pathogenicity tests carried out on detached leaves to verify Koch's postulates. Based on morphological observations and polygenic analyses, the pathogen causing leaf spots on S. nobilis was identified as Colletotrichum siamense.     

Interactions between callus cultures of Pinus sylvestris and pine fungi with different trophic properties

Fungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white‐creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25–35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis‐related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one‐third of the isolates of each fungus examined showed greater virulence compared to other isolates.     

Genotype distribution of Raffaelea quercivora in the oak galleries and its composition in the mycangia of Platypus quercivorus

Raffaelea quercivora is the pathogenic fungus that causes Japanese oak wilt. The female monogynous ambrosia beetle, Platypus quercivorus, carries this fungus in mycangia on the pronotum. These beetles bore galleries in oak trees with their partners to produce offspring, and they deposit fungus on the gallery walls from their mycangia. The offspring mature in the gallery, before loading the fungal pathogen and flying from the gallery to other healthy trees. To investigate the unloading and loading modes of the fungus within the gallery, we developed four polymorphic microsatellite markers for R. quercivora and identified the fungal genotypes in the galleries and mycangia of the beetles. Small wood chips were sampled at 5–10‐mm intervals from the walls of five galleries in a dead Quercus serrata tree. The pronota were also sampled from five female adult beetles. The genotypes of the R. quercivora isolates from the wood chips and pronota were identified using the microsatellite makers. The genotypic analysis showed that each gallery was inhabited patchily by 5–10 genotypes of R. quercivora, and the mycangia of each beetle contained 3–6 genotypes. These results indicate that diverse R. quercivora genotypes are unloaded repeatedly from the mycangia of female beetles onto the gallery wall, which results in their patchy distribution on the walls. When the offspring leave the host tree, the fungal clones that proliferate in the walls are also loaded repeatedly into the mycangia of the mature beetles.     

Pestalotiopsis biciliata,a new leaf pathogen of Eucalyptus spp. recorded in Italy

The present paper describes new symptoms on Eucalyptus spp. in Central Italy associated with the fungal pathogen Pestalotiopsis biciliata. This fungal species has been previously recorded in Italy on grape associated with fruit rot. Artificial inoculation trials on leaves confirmed the pathogenicity of the fungus to Eucalyptus camaldulensis. P. biciliata is included in the list of Pestalotiopsis spp. responsible for leaf disease on eucalypts worldwide.     

Higher resistance of the offspring of Scots pine trees resulting from natural regeneration in old foci of Heterobasidion annosum root rot

Abstract

Heterobasidion annosum (Fr.) Bref. is a fungal pathogen causing annosum root rot – one of the most economically important diseases in coniferous stands. The major aim of this study was to compare the resistance of the offspring of Scots pine trees (Pinus sylvestris L.) from seed orchards and the offspring of trees that were naturally reproduced in old foci of the disease. In experiments conducted in vitro, we used 960 seedlings which were grown from seeds collected from 60 trees in 6 old foci of the disease and 640 seedlings grown from seeds collected in 4 seed orchards. The offspring of trees from seed orchards after inoculation with H. annosum had nearly twofold higher mortality rate than the seedlings developed from the seeds collected in old foci of the disease. This suggests that the offspring of self-sown trees in old foci of the disease has a greater, genetically conditioned resistance to annosum root rot.     

Conifer species influence Heterobasidion irregulare sporulation on stem discs exposed to natural inoculum and root wood decay in soil‐block tests

The fungal pathogen Heterobasidion irregulare has caused substantial damage to pine species in much of the south‐eastern United States, but the potential for damage from Heterobasidion root disease to forest tree species of the Great Lakes Region has not been thoroughly investigated. In each of three trials in two infested stands, fresh stem discs of six native conifer species were exposed to natural inoculum and examined for the asexual Spiniger stage of H. irregulare after incubation in the laboratory. In the first trial, the fungus was infrequently observed on discs, and differences among species were not found. But in the other two trials, detection of the pathogen was much more frequent on discs of tamarack (Larix laricina), red pine (Pinus resinosa) and white spruce (Picea glauca) than those of northern white cedar (Thuja occidentalis), eastern hemlock (Tsuga canadensis) and balsam fir (Abies balsamea). In a second study, the ability of an isolate of H. irregulare to decay root sapwood of these same species was compared using a soil‐block decay test. After 16 weeks of incubation, weight loss due to decay was greatest for red pine (23.2%) and larch (19.1%) and least for balsam fir (1.3%). Differences in the ability of H. irregulare to infect and saprophytically utilize substrate of these important tree species should prompt additional investigation of the relative risk posed to forests of the Great Lakes Region.     

Shot hole disease on Prunus laurocerasus caused by Neofusicoccum parvum in Serbia

Between 2010 and 2014, symptoms of a shot hole disease were observed on cherry laurel (Prunus laurocerasus L.) trees and shrubs in parks and other public plantings in Belgrade, Serbia. Ten symptomatic leaves were collected from each of the diseased plants and the associated fungus isolated and identified using multigene phylogenetic analyses and asexual morphological characters. The pathogen was identified as Neofusicoccum parvum. The same symptoms were produced when the pathogen was inoculated on test plants. To the best of our knowledge, this is the first report of N. parvum causing shot hole disease on P. laurocerasus.     

Enhancing resistance in Pinus radiata seedlings to terminal crook (Colletotrichum acutatum) using methyl jasmonate and ultraviolet‐C radiation

Colletotrichum acutatum is a fungal pathogen that causes terminal crook disease in radiata pine (Pinus radiata) seedlings in New Zealand forest nurseries. Symptoms of infection include malformation or death of the growing tip and a stiffening and thickening of the stem. Although the disease can be managed effectively using fungicides, the New Zealand forest industry is interested in alternative control options such as induced resistance. The aim of this study was to evaluate spray application of chitosan (1.4 g/l) or 2.25 mm methyl jasmonate (MeJA) and irradiation with UV‐C (2.16 kJ/m²) for their potential to induce resistance to terminal crook. The treatments were applied to 4‐month‐old seedlings at 1 week before pathogen inoculation. By the end of the experimental period (42 days after inoculation), there was 80% disease incidence in the controls, with 48% of seedlings exhibiting severe terminal crook symptoms. The most effective treatment (p < 0.05) was MeJA with 16% disease incidence and none with severe symptoms. UV‐C also significantly (p < 0.05) reduced infection with 52% incidence and 20% of seedlings exhibiting severe symptoms. Chitosan did not reduce disease incidence (72%) compared with the control (80%) but did significantly reduce (p < 0.05) disease severity with 28% exhibiting severe symptoms. MeJA was the only treatment that significantly (p < 0.05) reduced the detrimental effects of infection on seedling apical growth and stem diameter. To our knowledge, this is the first report demonstrating the potential for MeJA and UV‐C to control terminal crook in radiata pine.     

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.     

Phylogenetic and pathogenicity evaluation of the marasmioid fungus Marasmius palmivorus causing fruit bunch rot disease of oil palm

Marasmius palmivorus is a marasmioid fungal species that exhibits parasitic behaviour, although most marasmioids are rarely parasitic. The fungus has been reported to cause fruit bunch rot disease of oil palm and coconut, but only a few studies on its pathogenic behaviour are available, particularly on oil palm. Hence, there is a need to assess the ability of the fungus to act as a pathogen and to study its molecular evolution and taxonomy. Nine isolates of M. palmivorus were successfully isolated from basidiocarps and diseased fruitlets of oil palm collected from oil palm plantations and were morphologically characterized on potato dextrose agar (PDA) followed by molecular identification based on nucleotide sequence alignments of internal transcribed spacer (ITS) regions of ribosomal RNA (rRNA) gene clusters with sequences from GenBank. Koch's Postulates confirmed that M. palmivorus could infect oil palm fruitlets with symptoms similar to those observed for bunch rot disease. Molecular phylogenetic studies using nucleotide sequences of ITS and the nuclear ribosomal large subunit (nLSU) showed that isolates sharing the same phenotypic characteristics of Marasmius palmivorus and Marasmiellus palmivorus are monophyletic and share a common ancestor. The fungus has also been shown to be more closely related to the genus Marasmius than Marasmiellus; therefore, we support retention of the taxon name of the pathogen causing bunch rot disease of oil palm as Marasmius palmivorus.     

Responses of swamp bay,Persea palustris,and avocado,Persea americana,to various concentrations of the laurel wilt pathogen,Raffaelea lauricola

Laurel wilt, caused by the fungus Raffaelea lauricola and transmitted by the exotic ambrosia beetle Xyleborus glabratus, has killed members of the Lauraceae plant family throughout the southeast United States. A series of experiments were conducted to examine the effects of inoculum concentration on the development of laurel wilt in swamp bay, Persea palustris, and avocado, Persea americana. In each experiment, host plants were inoculated with aqueous suspensions of 10², 10³, 10⁴ or 10⁵ conidia of R. lauricola, and plants were rated periodically for external symptom development (wilting and foliar dieback). At the end of experiments, plants were rated for internal symptoms of the disease (discoloration of sapwood) and assayed for R. lauricola on a semi‐selective medium. Symptom severity in swamp bay was significantly lower for the 10² treatment than at higher (10³–10⁵) concentrations, whereas 10² and 10³ conidia caused less disease than 10⁴ and 10⁵ conidia in avocado. At the lowest inoculum concentration, 67% of the swamp bay plants and 20% of the avocados died by the time the respective experiments were terminated. The pathogen was recovered from a high proportion of the symptomatic sapwood of swamp bay (100%) and avocado (94%), and sapwood discoloration and recovery of R. lauricola from inoculated stems of swamp bay were highly correlated with recovery of the pathogen and symptom development in roots. Clearly, swamp bay and avocado are very sensitive to R. lauricola. The ability of only 100 conidia of this pathogen to kill these hosts suggests that few individuals of X. glabratus or other ambrosia beetles that carry low levels of the pathogen would be sufficient to transmit conidia that infect and lead to disease development. The results are also relevant to the development of disease‐tolerant host selections, as they indicate levels of the pathogen appropriate for use in screening plants for disease resistance.     

Analysis of fungal endophytes in Scottish Sitka spruce plantations shows extensive infections,novel host partners and gives insights into origins

This preliminary study identified the fungal endophytes of Sitka spruce (Picea sitchensis) in four Scottish plantations, analysed their recruitment possibilities and community structure across the sites, and identified the possible origins of the endophytes in this non‐native host. Although Sitka spruce is native to north‐western North America, it comprises a huge portion of Scottish forestry and is an economically vital timber tree. Needles from two age classes were collected and cultured for emerging endophyte isolates. Using a combination of morphological features and genetic sequencing of the nuclear ribosomal ITS regions of representative morphological types, 57 morphotypes were recorded which represented at least 15 different species, including a hypothesized sister species to the pathogen of Pseudotsuga menziesii in North America and Europe, Nothophaeocryptopus gaeumannii (previously P. gaeumannii) that was present at all sites tested. Diversity, recruitment, site, needle age and needle part effects are discussed. The phylogeny of Nothophaeocryptopus based on analysis of combined ITS and Tub2 sequence data is given in order to assess the relationship of the unknown endophyte to the Douglas fir pathogen N. gaeumannii.     

Rapid detection of Fusarium circinatum propagules on trapped pine beetles

Pitch canker is a destructive disease of pine caused by the fungus Fusarium circinatum. This taxon is listed as a quarantine fungus for several regional plant protection organizations throughout the world. Whereas long‐distance spread of the disease is made possible through the trade of infected pine seeds, local spread is caused by aerial dispersion or insect transportation of the fungal conidia. Developing a reliable and efficient tool to detect of F. circinatum in insects would be very useful to monitor the local spread of the pathogen. This tool would also provide the means to assess the range of insect species that could serve as potential vector of the fungus. A DNA extraction protocol was optimized and combined with a real‐time PCR test to detect F. circinatum on pine beetles. Using artificially contaminated Ips sexdentatus, it was shown that the test was able to detect down to 10 F. circinatum conidia per individual, and 20 conidia per batch of 10 insects, which is below the lowest inoculum load occurring in nature. With this technique, several batches of up to 10 insects may be analysed simultaneously, with a timescale for analysis reduced to <5 h and without the need for expertise in Fusarium taxonomy. This tool may be useful to monitor potential spread of the pathogen across regions. Using this method, to date, despite F. circinatum foci occurred in Northern Spanish regions across the border in France, the pathogen was not found on I. sexdentatus.     

qPCR quantification of Ophiognomonia clavigignenti‐juglandacearum from infected butternut trees under different release treatments

The use of a molecular assay for quantifying conidia of Ophiognomonia clavigignenti‐juglandacearum, the fungal pathogen responsible of butternut canker, was investigated. Purified DNA from conidia collected on glass fibre filters of a passive rain collectors was quantified using a TaqMan real‐time quantitative polymerase chain reaction (qPCR) assay. The qPCR assay could specifically discriminate the target species from all other North American known species of Ophiognomonia, and it was sensitive enough to repeatedly detect one conidium. A linear relationship between numbers of conidia and qPCR C_t values was determined, and used to assess the sporulation of the pathogen under trees that were released to promote their vigour. In total, 977 samples of field‐captured conidia from 49 trees, at two locations, and from two successive growing seasons were analysed. No significant difference of sporulation was observed under control and release treatments. However, our results demonstrated that qPCR assay was reliable for detecting and quantifying O. clavigignenti‐juglandacearum from environmental samples, which will be useful to assess further control methods for this disease.