First record of Ektaphelenchoides pini associated with Ips sexdentatus on Pinus nigra laricio in Italy

In February 2015, an unexpected windstorm downed five hectares of a European black pine Pinus nigra subsp. laricio forest formation located close to Vallombrosa, Florence (Central Italy). In the following spring, an extensive survey was conducted in the area. Felled trees, stumps and all the suitable plant material were screened for the presence of the pinewood nematode (PWN), Bursaphelenchus xylophilus, by sampling wood and bark. Bark beetles were then collected from the gallery systems on the inner side of bark samples and observed in the laboratory. The following bark beetles were morphologically identified: Ips sexdentatus, Orthotomicus erosus, O. laricis and Pityogenes bidentatusa. The dissection of Ips sexdentatus allowed the extraction of numerous nematodes that were morphologically and molecularly identified as Ektaphelenchoides pini. Conversely, only few nematode specimens were isolated from either pine bark or wood. These individuals could be only molecularly identified and belonged to an undescribed nematode taxon. Even though no PWN was recorded in the investigated sites, our survey allowed the detection of a new association between E. pini and I. sexdentatus on P. nigra.     

First molecular detection of Lecanosticta acicola from Poland on Pinus mugo

The presence of quarantine pathogen Lecanosticta acicola, the causal agent of brown spot needle blight, was confirmed using molecular methods for the first time in northern part of Poland on the coastal area adjacent to the Baltic Sea. This area includes sandy beaches, where Pinus mugo and P. sylvestris were planted. Symptomatic needles were collected in 2017 from 20 P. mugo trees growing in one stand in Ustka. Typical symptoms of brown spot needle blight infection, including dead needle tips and central zones with yellow or reddish brown, circular spots in green tissue, were observed on all samples. Only, the asexual stage of L. acicola was obtained during this work. The pathogen species identity was confirmed using classical morphological methods (microscopic examination of the infected needles), real‐time and species‐specific priming (SSPP) PCR, and ITS sequencing. Analysis of mating‐type (MAT) genes showed the presence of both mating types in northern Poland.     

Genotypic diversity and distribution of Sphaeropsis sapinea within Pinus radiata trees from northern Spain

Sphaeropsis sapinea is an important latent pathogen of Pinus spp., outbreaks of which have a considerable impact on plantations. This study considers the population diversity and distribution of S. sapinea in northern Spain at different spatial scales from single plantations to a wide area covered by Pinus radiata trees. Estimation of genotypic diversity is an important component of the analysis of the genetic structure of plant pathogen populations. Ten simple sequence repeat (SSR) markers were used, together with vegetative compatibility tests, to study the genetic diversity among S. sapinea isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of S. sapinea isolates. From a total of 86 isolates collected from four different areas, 14 microsatellite haplotypes and 13 vegetative compatibility groups (VCGs) were identified. The percentage of maximum genotypic diversity, based on Stoddart and Taylor's index, for microsatellites of the northern Spain population ranged from 14.6% to 38.1% and from 8.0% to 29.4% for VCGs. Analysis of these markers and vegetative compatibility groups confirmed that S. sapinea reproduces mainly asexually due to its reduced genotypic diversity in spatially close populations. Isolates of S. sapinea from northern Spain populations were predominantly monomorphic at the tested SSR loci. Vegetative compatibility groups also indicate a low level of genetic variability in these samples, which appear to be clonal.     

First detection of Neoscytalidium dimidiatum associated with canker disease in Egyptian Ficus trees

In September 2013, a canker disease of Ficus nitida and F. benjamina was reported for the first time in Assiut governorate, Egypt. Infected samples were collected from various locations. Pure cultures of a fungus were isolated on potato dextrose agar at 25°C from diseased plants. Morphological investigation and DNA sequencing showed that the causal agent was Neoscytalidium dimidiatum. A pathogenicity test conducted using 2‐year‐old plants of each host gave 70%–80% infection, and the pathogen was reisolated from the inoculated plants. A pure culture of N. dimidiatum was deposited in the culture collection of the Assiut University Mycological Centre (AUMC) under the code AUMC 9293 and the ITS sequence was placed in NCBI under accession number KX985929 .     

First report of Lecanosticta acicola on non‐native Pinus mugo in southern Sweden

In 2017, severe symptoms of brown spot needle blight, similar to those caused by Lecanosticta acicola, were observed on needles of non‐native Pinus mugo var. Hesse planted in an arboretum in southern Sweden. Microscopic characterization and molecular diagnostics of isolates obtained from diseased needles confirmed the identity of L. acicola. This is the first report of the quarantine pathogen L. acicola in Sweden. Subsequent surveys are urgently needed to assess the presence and extent of the L. acicola invasion in Scots pine (Pinus sylvestris) stands in Sweden and other Scandinavian countries.     

Life cycle and in vitro sporulation dynamics of Corinectria constricta,the causal agent of Pinus radiata stem canker,in Chile

In 2008, a canker disease caused by the fungus Corinectria constricta was detected in southern Chile. The causal agent was previously identified as Neonectria fuckeliana (now Corinectria fuckeliana), which has been associated with stem cankers in Pinus radiata plantations in New Zealand since the 1990s. Many basic aspects of the life cycle of C. constricta remain unknown. The current study aimed to (a) document the periods during which C. constricta fruiting bodies are present in P. radiata plantations and associated factors; (b) determine the C. constricta life cycle in P. radiata plantations in southern Chile; and (c) evaluate, under in vitro conditions, the sporulation dynamics of ascospores. The first and second aims were carried out by evaluating affected plantations every 15 days, identifying asexual and sexual fungal structures, and recording the time periods when the structures were present. The third aim was achieved with in vitro tests in Petri dishes simulating humidity chambers. The life cycle was characterized by the presence of sporodochia from the Cylindrocarpon‐like (asexual form of C. constricta) morph during the autumn of 2012 (March–May). Subsequently, perithecia began to form on the sporodochia during April of 2012, taking approximately 3 months to mature (May–July), persisting for the rest of the year and providing inoculum to infect new trees. The development of perithecia in winter demonstrates that this is the most important period for dispersal and infection. In terms of sporulation dynamics, perithecia can release ascospores up to eight days following a wetting event; without this event, the spores are not released.     

First report of Lasiodiplodia theobromae and Fusarium proliferatum causing teak die back in Costa Rica

Teak plantation productivity and economic value have risen significantly since 2000; however, teak dieback syndrome in commercial plantations is becoming worse every year. We isolated, characterized and identified the causal agents of teak dieback, based on morphological characteristics in culture and DNA sequencing. The pathogenicity of Lasiodiplodia theobromae and Fusarium proliferatum (separate and together) was confirmed in inoculations of teak elite genotypes, fulfilling Koch's postulates. To our knowledge, this is the first report of L. theobromae and F. proliferatumcausing teak dieback syndrome in Costa Rica.     

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.     

Host specificity of co‐infecting Botryosphaeriaceae on ornamental and forest trees in the Western Balkans

The Botryosphaeriaceae is a diverse family of endophytes and fungal pathogens of mainly woody plants. We considered the host range and distribution of these fungi by sampling diseased ornamental and forest trees and shrubs in Serbia, Montenegro, Bosnia and Herzegovina, spanning a Mediterranean and a Continental climatic region. In total, ten Botryosphaeriaceae species were identified in the Western Balkans and with the exception of Sphaeropsis visci and Phaeobotryon cupressi, which occurred on one host, all the species had a broader host range. Phaeobotryon cupressi was found only in the Mediterranean region and S. visci, Dothiorella sp., Dothiorella sarmentorum and Diplodia seriata were present only in the Continental region. Pathogenicity tests were conducted on a variety of hosts from which the Botryosphaeriaceae species were isolated. These included leaves and/or stems of seedlings of 21 hosts, and cut leaves and/or branches of six hosts. Moreover, stems of seedlings of Chamaecyparis lawsoniana, Cedrus deodara, Picea omorika, Pinus patula and Eucalyptus grandis were inoculated as hosts from which some or all of the Botryosphaeriaceae species used for inoculation were not isolated. Inoculations showed that the majority of these fungi could also co‐infect hosts other than those from which they were isolated. The results suggest that most of the species have broad host ranges and can potentially cause disease on a broad range of tree species under certain conditions.     

Identification and characterization of bacterial strains associated with diseased oak trees in Northern Iran

Oak decline syndrome is characterized by periodic occurrences of decline and death of oaks over widespread areas. An outbreak of a new emerging disease on oak trees was reported in the Hyrcanian forest of Iran (Mazandaran and Golestan provinces) that showed stem bleeding and canker symptoms. Bacterial isolates were characterized through biochemical and physiological tests, protein electrophoresis, DNA fingerprinting (rep‐PCR, ERIC and BOX primers) and sequencing of 16S rRNA and MLSA (multilocus sequencing analysis) for housekeeping genes (gyrB, infB and atpD). A complex community of the genus Brenneria spp. (Brenneria goodwinii, Brenneria roseae subsp. roseae, Brenneria sp. and Brenneria nigrifluens) and a few isolates in the genus Gibbsiella were identified as major groups involved. Isolate differentiation was more accurate using concatenated partial gene sequences within the main groups. All bacterial isolates showed hypersensitivity reactions (HR) on Pelargonium leaves (Pelargonium × hortorum). Pathogenicity studies of different Brenneria and Gibbsiella strains revealed that they have potential to cause the disease in oak seedlings and devastating oak canker and stem bleeding symptoms in northern Iran. Due to the presence of several potentially pathogenic agent(s) associated with the oak decline, identification of the principal agent(s) is of major interest. To our knowledge, this is the first report of potentially pathogenic bacteria associated with oak bleeding and canker in Iran.     

Effects of irrigation on water use and water use efficiency in two fast growing Eucalyptus plantations

Eucalyptus plantations occupy almost 20 million ha worldwide and exceed 3.7 million ha in Brazil alone. Improved genetics and silviculture have led to as much as a three-fold increase in productivity in Eucalyptus plantations in Brazil and the large land area occupied by these highly productive ecosystems raises concern over their effect on local water supplies. As part of the Brazil Potential Productivity Project, we measured water use of Eucalyptus grandis × urophylla clones in rainfed and irrigated stands in two plantations differing in productivity. The Aracruz (lower productivity) site is located in the state of Espirito Santo and the Veracel (higher productivity) site in Bahia state. At each plantation, we measured stand water use using homemade sap flow sensors and a calibration curve using the clones and probes we utilized in the study. We also quantified changes in growth, leaf area and water use efficiency (the amount of wood produced per unit of water transpired). Measurements were conducted for 1 year during 2005 at Aracruz and from August through December 2005 at Veracel. Transpiration at both sites was high compared to other studies but annual estimates at Aracruz for the rainfed treatment compared well with a process model calibrated for the Aracruz site (within 10%). Annual water use at Aracruz was 1394 mm in rainfed treatments versus 1779 mm in irrigated treatments and accounted for approximately 67% and 58% of annual precipitation and irrigation inputs respectively. Increased water use in the irrigated stands at Aracruz was associated with higher sapwood area, leaf area index and transpiration per unit leaf area but there was no difference in the response of canopy conductance with air saturation deficit between treatments. Water use efficiency at the Aracruz site was also not influenced by irrigation and was similar to the rainfed treatment. During the period of overlapping measurements, the response to irrigation treatments at the more productive Veracel site was similar to Aracruz. Stand water use at the Veracel site totaled 975 mm and 1102 mm in rainfed and irrigated treatments during the 5-month measurement period respectively. Irrigated stands at Veracel also had higher leaf area with no difference in the response of canopy conductance with air saturation deficit between treatments. Water use efficiency was also unaffected by irrigation at Veracel. Results from this and other studies suggest that improved resource availability does not negatively impact water use efficiency but increased productivity of these plantations is associated with higher water use and should be given consideration during plantation management decision making processes aimed at increasing productivity.     

Rapid detection and identification of ‘Candidatus Phytoplasma pini’‐related strains based on genomic markers present in 16S rRNA and tuf genes

In order to devise a method for rapid detection of ‘Candidatus (Ca.) Phytoplasma pini’ and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian ‘Ca. Phytoplasma pini’ strain PineBL2 using Illumina (NGS) technology and targeted sequencing employing universal phytoplasma primers. We focused on two resulting chromosomal segments that contained a 16S rRNA gene and a translation elongation factor EF‐TU gene (tuf), respectively. Based on alignments of the ‘Ca. Phytoplasma pini’ gene sequences with the corresponding sequences of other phytoplasmas, we designed new primer pairs for PCR‐based detection of ‘Ca. Phytoplasma pini’. Because ‘Ca. Phytoplasma pini’ strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and PCR protocols designed in the current work enabled rapid direct PCR detection and identification of ‘Ca. Phytoplasma pini ’ by amplifying a 484 bp 16S rDNA segment and a 513 bp tuf gene fragment that contain regions unique to this phytoplasma .     

Spatiotemporal analysis of pine wilt disease: Relationship between pinewood nematode distribution and defence response in Pinus thunbergii seedlings

Pine wilt disease is of major concern as it has destroyed pine forests in East Asia and Europe. Several studies have suggested that invasion by the pinewood nematode (PWN) Bursaphelenchus xylophilus, which causes this disease, evokes an excessive defence response in pine trees, resulting in tree death. However, few studies have quantitatively evaluated the correlation between PWN distribution and tree defence responses. Therefore, the present study aimed to quantify the number of PWNs and expression levels of putative pathogenesis‐related (PR) genes in different positions of Japanese black pine (Pinus thunbergii) seedlings over time. To quantify the number of PWNs in the seedlings, we used TaqMan quantitative real‐time PCR (qPCR) assay. During the early phase of infection, most PWNs were distributed around the inoculated sites, with only a small number being detected at distant sites, but the expression levels of PR genes were highly upregulated throughout the seedlings. Both the number of PWNs and expression levels of PR genes then increased drastically throughout the seedlings, all of which exhibited external symptoms. Thus, it appears that the rapid migration of PWNs induces a defence response throughout the seedling; however, this may not be effective in controlling these parasites, thereby ultimately leading to plant death.