Cryphonectria hypovirus 1 (CHV‐1) survey reveals low occurrence and diversity of subtypes in NE Spain

Cryphonectria parasitica (Murrill.) Barr is a necrotrophic fungus that affects European chestnut populations. In Catalonia (NE Spain), this fungus is widely spread affecting chestnut stands. In addition to vegetative compatibility (VC) types of the fungus, knowledge of the Cryphonectria hypovirus (CHV) occurrence is important to implement a biocontrol programme. We confirmed the presence of hypoviruses in several chestnut subpopulations of Catalonia, and we determined the VC types of CHV‐infected isolates. We also studied the nucleotide sequences of these hypoviruses from a variable region located in the hypovirus ORF‐A. Our results show a low occurrence of hypoviruses throughout C. parasitica populations in Catalonia, except for a few localized stands. From 312 sampled cankers, we obtained 179 white, pale orange or rickety isolates. In 35 of them, we detected CHV‐1. Infected isolates belonged to 5 of the 7 sampled subpopulations. We found 12 CHV‐1 haplotypes, based on the nucleotide sequence analysis. Most of the hypoviruses have the same nucleotide sequence or show high homology at nucleotide level with isolates previously included in CHV‐1 subtype‐I. However, we found that 3 haplotypes share at least 97% of their ORF‐A nucleotide sequence with CHV‐1 subtype‐E. Phylogenetic analysis grouped these 3 isolates in a different cluster than the other hypoviruses. These results suggest that multiple introductions of CHV‐1 have occurred recently in Catalonia. Dominant VC types of hypovirus‐infected isolates are EU‐1, EU‐2 and EU‐5, which are similar to those previously reported in this region.     

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.     

Update on the 35‐year expansion of the invasive root pathogen,Phytophthora lateralis,across a landscape of Port Orford cedar (Chamaecyparis lawsoniana)

Port Orford cedar (Chamaecyparis lawsoniana) is endemic to northern California and southwestern Oregon and is considered a foundation species that plays critical roles in riparian areas and on nutrient‐poor soils. Since 1952, a non‐native, pathogenic oomycete (Phytophthora lateralis) has been spreading throughout the range of the cedar. Most spread occurs by vehicles carrying infested soil along gravel roads primarily used for timber harvest. In a previous study conducted in 1998 and 1999, Port Orford cedar and P. lateralis were censused in a 37‐km² study area and dendrochronology was used to reconstruct the history of pathogen invasion. That work, which represents the only detailed analysis of spread rates for P. lateralis, showed that the first successful invasion into the study area took place in 1977 and that 43% of the susceptible host sites (stream crossings) were infested by 1999. In the work presented here, all sites that were uninfested in 1999 were re‐censused in 2012, extending the historical reconstruction of P. lateralis spread to 35 years. Two new infestations were initiated between 1999 and 2012, suggesting that the rate of spread of P. lateralis has slowed greatly. Between 1980 and 1989, the average number of new site infestations was 1.8 infestations per year, while between 1990 and 1999 the average was 0.4 infestations per year and between 2000 and 2009 the average was 0.2 infestations per year. Several potential explanations for the reduced number of new infestations are discussed.     

Genetic variation in dieback resistance in Fraxinus excelsior confirmed by progeny inoculation assay

Ash dieback caused by the pathogenic fungus Hymenoscyphus fraxineus [previously known as H. pseudoalbidus (sexual stage) and Chalara fraxinea (asexual stage)] is a widespread problem in Europe. Here, we assess crown damage from natural infection and necrosis development following artificial controlled inoculations on full‐sib and half‐sib progeny from Danish Fraxinus excelsior clones with contrasting and well‐characterized levels of susceptibility to the disease. The inoculation assay was performed on a total of 123 offspring, and necrosis development monitored over two years. The offspring from low susceptible mother clones developed smaller necroses when compared to offspring from susceptible clones. Their crown damage due to natural infections was also significantly less. The correlation coefficient between average crown damages of mother clones and the average of their progeny was 0.85 (natural infections), while the correlation between crown damage of mother clones and the average necrosis development in their progeny after controlled inoculation was 0.73. The correlation between resistance of parent trees and crown damage/necrosis development on their offspring confirms the presence of heritable resistance and indicates that a bioassay based on controlled inoculations has the potential of becoming a fast and cost‐effective tool for estimation of dieback susceptibility in breeding programmes for resistance in ash trees.     

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.     

Establishment and dissemination of hypovirulent strains of Cryphonectria parasitica in Greece

Chestnut blight caused by Cryphonectria parasitica has been one of the major reasons for the decline of chestnut cultivation in Greece over the last 50 years. A previous detailed study of the vc types of the fungus has revealed only four vc types in the entire country, those of EU‐1, EU‐2 and EU‐10 with the dominant being EU‐12 counting for 88% of the isolates. As the loss in orchard trees reached in some chestnut producing prefectures over 35% of the trees, the decision was taken to apply biological control on a nationwide scale. The project was implemented in 17 prefectures during the period 2007–2009. Hypovirulent (hv) strains of C. parasitica which were infected by the CHV‐1 subtype I (Italian subtype) viruses which occur naturally in Greece were used. The necessary hypovirulent inoculum was prepared at the Forest Research Institute during the period 2007–2009 on a large scale, however, with laboratory care. Three million inoculations were made around accessible developing cankers over three consecutive years by trained personnel. An extensive evaluation conducted in 2011 in the 12 of the 17 treated prefectures, where no natural hypovirulence had been found before, demonstrated not only the successful establishment of hypovirulence but also profound dissemination and healing of non‐inoculated cankers. The extent of dissemination varied significantly among sampling plots and among prefectures. The conclusion is drawn that CHV‐1 subtype I has successfully established and spread into chestnut orchards and coppice forests resulting in a gradual decline of chestnut blight.     

Large‐scale fuzzy rule‐based prediction for suitable chestnut ink disease sites: a case study in north‐east Italy

In the past few decades, economic interest in the cultivation of chestnuts for both timber and nut production has resurfaced in the Mediterranean area. However, chestnut cultivation has suffered in recent years from the spread of exotic pests, such as the gall wasp Dryocosmus kuriphilus, and from the resurgence of previously present diseases, most likely due to anomalous climate dynamics. This is the case with chestnut ink disease, caused by the soilborne pathogens Phytophthora cinnamomi and P. cambivora. Scientific and technical support in monitoring and management, that utilizes new forecasting approaches incorporating related environmental variables, is therefore essential. The main aim of this study was to develop a mathematical model assessing the potential for the establishment of chestnut ink disease at a large scale. Towards this goal, fuzzy rule‐based theory was applied to the environmental variables associated with host presence, pathogens' ecological niches and ink disease symptoms expression. The effectiveness of the rule‐based modelling outcomes, provided with uncertainty maps to facilitate their correct interpretation, was confirmed by detailed field data collected from a large chestnut‐growing area where ink disease has been increasing in recent years. The final model gave consistent predictions for disease presence. For this reason, it represents a flexible and valuable decision‐support tool to forecast which sites are at risk from CID.     

Variation in pathogenicity among the three subspecies of Phytophthora alni on detached leaves,twigs and branches of Alnus glutinosa

Pathogenicity tests were carried out on leaves, twigs and branches of Alnus glutinosa using several isolates of Phytophthora alni ssp. alni, P. alni ssp. multiformis and P. alni ssp. uniformis in vitro. Healthy fresh leaves were collected from disease‐free areas and inoculated with mycelium on agar discs or by dipping in zoospore suspensions. In addition, twigs and branches were collected from both disease‐free and disease‐affected areas, inoculated with mycelium on agar discs and incubated at four temperatures (15, 20, 25, 30°C). All subspecies tested were pathogenic but with varied level of virulence. In inoculation tests on foliage, wounding was a key factor in causing infections: lesions on inoculated wounded leaves were larger than on non‐wounded leaves. In the twig and branch inoculation tests, no differences in virulence were observed among the P. alni subspecies in terms of sampling locations, but lesions differed in size according to incubation temperature, with the largest lesions occurring on tissues incubated at 25°C. The work is the first to report foliar necrosis caused by P. alni on A. glutinosa. P. alni ssp. uniformis was the least virulent of the subspecies in branch inoculations. These findings demonstrate that various tissues of A. glutinosa could act as sources of pathogen inoculum and may disseminate alder Phytophthora in natural ecosystems.     

First definite report of natural infection of Fraxinus ornus by Hymenoscyphus fraxineus

Hymenoscyphus fraxineus was isolated from four leaf rachises with necrotic lesions of flowering ash (Fraxinus ornus) seedlings, which had been planted on a forest site in Austria where they were exposed to a massive natural infection pressure. This represents the first definite report of natural infection of this ash species by the ash dieback pathogen.