Endochitinase activity in the apoplastic fluid of Phellinus weirii‐infected Douglas‐fir and its association with over wintering and antifreeze activity

Extracellular proteins were extracted from Phellinus weirii infected Douglas‐fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase‐like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two‐dimensional gel electrophoresis (2‐D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27–30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii‐infected winter Douglas‐fir needles showed anti‐freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response.     

Occurrence of Phytophthora cinnamomi in cork oak forests in Italy

An increasing decline and mortality of cork oak trees have been recently observed in central Italy and Sardinia Island. Following surveys conducted in three declining cork oak forests, a Phytophthora species was consistently isolated from soil samples collected from trees displaying different level of decline. Based on morphological features, growth rates at different temperatures and analysis of DNA sequences of the ITS region, all isolates were identified as Phytophthora cinnamomi Rands. This pathogen caused large brownish lesions on inoculated freshly cut branches of cork oak. It was re‐isolated from all infected tissues. These findings represent the first report of P. cinnamomi on cork oak trees in Italy.     

First report of Phytophthora cinnamomi in forest stands in Germany

Phytophthora cinnamomi was repeatedly isolated from the rhizosphere of recently dead sweet chestnut trees in the upper Rhine valley. Identification was genetically confirmed by RFLP and sequencing of the ITS‐region. This is the first record of P. cinnamomi on forest land in Germany. Mode of introduction of this invasive species and potential factors favouring establishment are discussed.     

Development and use of new sensitive molecular tools for diagnosis and detection of Melampsora rusts on cultivated poplar

Poplar rusts due to Melampsora larici‐populina (Mlp), M. allii‐populina (Map) and M. medusae f. sp. deltoidae (Mmd) are the most serious disease in Europe on cultivated poplars, that is, Populus × euramericana and P. × interamericana hybrids. These pathogenic species can be identified by the observation of morphological characteristics of urediniospores but this method is not appropriate for high‐throughput analysis and cannot be used on other spore stages, such as aeciospores or teliospores, that are morphologically similar. The aim of this study was to develop a rapid and sensitive molecular method based on PCR amplification that was able to specifically detect these species on various hosts for routine analysis. Three primer pairs ITS‐MLP‐F/ITS‐MLP‐R, ITS‐MAP‐F/ITS‐MAP‐R and ITS‐MMD‐F/ITS‐MMD‐R were designed within the internal transcribed spacer (ITS) sequences of ribosomal DNA to target Mlp, Map and Mmd, respectively, and their specificity were confirmed on a wide range of isolates and species. ITS‐MLP‐F/ITS‐MLP‐R and ITS‐MAP‐F/ITS‐MAP‐R primers proved to be highly specific to Mlp and Map, respectively, whereas ITS‐MMD‐F/ITS‐MMD‐R cross‐reacted with DNA from M. larici‐tremulae and M. pinitorqua. However, these species are not pathogenic on cultivated poplars that all belong to sections Aigeiros and Tacamahaca of the genus Populus. Specific Mmd primers proved to be very sensitive as a positive signal could be obtained with DNA extracts from 6 target urediniospores mixed with 800 000 urediniospores of Mlp. An internal amplification control (IAC) was included to discriminate false negative results due to the potential presence of inhibitory compounds in DNA extracts. ITS‐MMD‐F/ITS‐MMD‐R primers are therefore efficient for the detection of the quarantine pathogen Mmd on samples collected on poplar or larch and are fit for use in official tests. This new PCR assay has been used in routine for ten years, and Mmd has hitherto never been detected in commercial poplar nurseries in France.     

Pathogenicity testing of four Bursaphelenchus species on conifer seedlings under greenhouse conditions

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, J. Agric Res. 48, 1934, 949), Nickle (J. Nematol. 2, 1970, 375), is the causative agent of the pine wilt disease and causes serious damage to pine forests around the world. During a survey for the pinewood nematode, four other Bursaphelenchus species (Bursaphelenchus mucronatus, B. sexdentati, B. anamurius and B. vallesianus) were isolated from wilted pine trees in Turkey. To understand the effects of these Bursaphelenchus species on wilting of pine trees, a study was conducted under greenhouse conditions. Two‐year‐old seedlings of three pine species (Pinus nigra, P. brutia and P. pinea) and one cedar species (Cedrus libani) were used. Fifteen seedlings of each species were inoculated with nematodes and 10 seedlings of each species served as controls. The inoculum densities used for each seedling contained approximately 1000 (±100) nematodes of all life stages in 0.25 ml of distilled water. The first wilting symptoms were observed in the fifth week in all pine species but not in the cedar seedlings. All seeding mortality occurred between the 5th and 13th weeks of the study; no mortality was observed outside of this period. The most pathogenic nematode species was B. mucronatus, closely followed by the other species. The most susceptible seedling species was P. nigra, and C. libani was the most resistant species.     

Relationships of stem surface area to other stem dimensions for Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don) and Japanese cypress (<Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> Endl.) trees

I investigated the relationships between stem surface area outside bark and other stem dimensions for Japanese cedar (Cryptomeria japonica D. Don) and Japanese cypress (Chamaecyparis obtusa Endl.) trees. The stem dimensions used here were the basal area and the product of diameter at breast height (dbh) and total tree height. The regression equation of the stem surface area s against basal area g was s = 184.216g for the cedar trees and s = 156.878g for the cypress trees. The slope of the equation was significantly different between the two species. For the same dbh, the cedar trees tended to have a higher total tree height than the cypress trees. The cedar trees also had a larger surface area of relative stem form than the cypress trees. These results indicated that the difference in the slope was produced by the differences in both the stem slenderness and tapering between the two species. On the other hand, the regression equation between the stem surface area and the product of dbh and total tree height dh was s = 1.937dh for the cedar trees and s = 1.921dh for the cypress trees, whereas no significant difference in the slope was found. The obtained slopes for the cedar and cypress trees seemed to be in accord with that for other coniferous species reported in earlier studies, suggesting that the variation in the slope among coniferous species would be small. The estimation from the basal area would provide a simpler means for estimating the stem surface area and would be useful in obtaining an approximation of the surface area. By contrast, the estimation from the product of dbh and total tree height would provide a more accurate and precise estimate as well as a wider applicable range, i.e., a parameter for physiological growth models. In conclusion, it could not be judged which regression equation examined in the present study was superior to the other, and thus it was important to select an appropriate equation depending both on the purpose and on the time and labor available.     

Rapid diagnosis of pathogenic Phytophthora species in soil by real‐time PCR

Real‐time PCR assays based on the TaqMan system and using ITS sequences were developed for the identification of Phytophthora species, including P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina, all of which are currently causing significant damage to roots of forest trees in both managed stands and natural ecosystems. Total genomic DNA was extracted from mycelia of aforementioned Phytophthora isolates. Species‐specific primers for P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina were designed based on ITS sequences of rDNA. The amplification efficiency of target DNA varied from 93.1% (P. pseudosyringae) to 106.8% (P. quercina). The limit of the detection was calculated as 100 – 1,000 fg DNA, depending on the Phytophthora species. In mixed soil samples, all Phytophthora species were detected for Ct values shifted by 0.7 – 2.1 cycles. Based on these real‐time PCR assays we were able to identify the five Phytophthora species. These techniques will be of value in the identification of these pathogens, which may cause up to 80 – 90% fine root loss in oak stands.     

First report of Cryptosphaeria pullmanensis as causal agent of Cryptosphaeria canker of Populus nigra in Iran

A diatrypaceous fungus was isolated consistently from cankers on Populus nigra trees showing dieback symptoms in Kohgiluyeh Boyer‐Ahmad, Zanjan and Esfahan provinces in Iran. Morphological characteristics and phylogenetic analyses of the ITS region of the rDNA identified the taxon as Cryptosphaeria pullmanensis. Pathogenicity tests conducted in potted 3‐month‐old cuttings of P. nigra confirmed Koch's postulates and revealed that C. pullmanensis caused canker on this host. This is the first report of C. pullmanensis causing a canker disease on P. nigra in Iran.     

Characterization of Corynelia uberata Fr., a putative fungal pathogen of Podocarpus falcatus in Ethiopian forests

Corynelia spp. are ascomycetes belonging to the order Coryneliales and are thought to be obligate parasites of trees in the Podocarpaceae. The aims of this study were to determine the disease intensity of Corynelia infection on Podocarpus falcatus in Ethiopian forests and verify the identity of Corynelia spp. from Ethiopia and other countries using morphological and molecular methods. Disease surveys were conducted in P. falcatus forest areas at Adaba‐Dodola, Bushoftu, Menagesha, Shashamane and Wondo Genet in Ethiopia between 2009 and 2011, and samples were collected for morphological and molecular studies. Additional dried specimens morphologically collected as C. uberata, C. portoricensis and C. tropica from Podocarpus species in Kenya, South Africa, Puerto Rico and New Zealand were also characterized. Morphologically, the South African specimen (F‐006479) of C. uberata had significantly larger ascospores when compared with the other specimens. There was a high sequence similarity (99–100%) in the internal transcribed spacer and 5.8S (ITS‐5.8S) region among the studied C. uberata sequences. Cloning and amplification of the insert spanning partial small ribosomal unit (SSU) and ITS‐5.8S regions of ribosomal DNA validated the unidentified ITS‐5.8S region as the sequence of C. uberata by inferring the reference sequence of SSU rDNA of C. uberata in GenBank. Both neighbour‐joining and/or maximum parsimony methods placed ITS‐5.8S and SSU rDNA sequences of Corynelia spp. at the basal position of the clade Eurotiomycetidae. C. uberata was found to be a potential pathogen on leaves, fruits and young stems of P. falcatus in Ethiopia.     

Characterization,pathogenicity and host range studies of Paecilomyces formosus associated with dieback of Christ's thorn trees (Paliurus spina-christi Mill.) in Iran

Christ's thorn (Paliurus spina-christi Mill.) tree is an important and valuable forestry species and has significant potential in afforestation and reforestation systems. In recent years, dieback symptoms were observed on Paliurus spina-christi trees in Gilan-e Gharb region of Kermanshah province, west Iran. During 2020 to 2021, Christ's thorn trees that exhibit branch dieback symptoms were sampled for fungal isolations. The pathogenicity tests, biochemical assay of growth, acid production on creatine sucrose agar (CREA) medium and morphological and molecular investigations (ITS and β-tubulin regions) identified Paecilomyces formosus as a dieback causal agent. This is the first report of P. formosus pathogenicity on Paliurus spina-christi trees. Furthermore, disease severity was assayed on 11 tree species under three different temperatures 15, 25 and 35°C. Disease severity caused by all isolates on detached branches was greater at 35°C than at 15 and 25°C. This study presents the host range of this pathogen and showed that these potential hosts are prone to this pathogen under high temperatures, which forest trees experienced in recent decades.     

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.     

Multispecies Phytophthora disease patterns in declining beech stands

Decline diseases are typically caused by complex abiotic and biotic interactions and characterized by a suite of symptoms indicative of low plant vigour. Diseased trees are frequently infected by Phytophthora, but the complex interactions between pathogen, host and the heterogeneous forest environment mask a comprehensive understanding of the aetiology. In the present study, we surveyed European beech (Fagus sylvatica) stands in Swiss forests with recent increases in bleeding lesions for the presence of Phytophthora. We used a combined approach of analysing soil and bark samples from trees displaying bleeding lesions and trees free from bleeding lesions. Soil baiting revealed a higher prevalence of Phytophthora spp. around trees with bleeding lesions than around trees without bleeding lesions. For the bark samples from bleeding lesions, we used several detection methods. Phytophthora spp. were detected in 74% of the trees by an immunological on‐site diagnostic kit, in 64% by a specific PCR assay, and 38% by isolation on selective media. All samples tested were negative for P. ramorum using qPCR. Overall, nine Phytophthora species were identified by ITS sequencing, the most common of which were P. plurivora, P. gonapodyides, P. × cambivora and P. syringae. We identified distinct species in bleeding lesions and the rhizosphere of the same host tree which suggests a multispecies Phytophthora disease patterns in these declining beech. Among the recovered species, P. × cambivora and P. × serendipita were identified as hybrid genotypes with the former abundant in bleeding lesions.     

Botryosphaeriaceae associated with die‐back of Schizolobium parahyba trees in South Africa and Ecuador

Die‐back of Schizolobium parahyba var. amazonicum is a serious problem in plantations of these trees in Ecuador. Similar symptoms have also been observed on trees of this species in various parts of South Africa. The most common fungi isolated from disease symptoms on S. parahyba var. amazonicum in both locations were species of the Botryosphaeriaceae. The aim of this study was to identify these fungi from both Ecuador and South Africa, and to test their pathogenicity in greenhouse and field trials. Isolates obtained were grouped based on culture morphology and identified using comparisons of DNA sequence data for the internal transcribed spacer (ITS) and translation elongation factor 1α (TEF‐1α) gene regions. The β‐tubulin‐2 (BT2) locus was also sequenced for some isolates where identification was difficult. Three greenhouse trials were conducted in South Africa along with a field trial in Ecuador. Neofusicoccum parvum was obtained from trees in both areas and was the dominant taxon in South Africa. Lasiodiplodia theobromae was the dominant taxon in Ecuador, probably due to the subtropical climate in the area. Isolates of Neofusicoccum vitifusiforme (from South Africa only), Neofusicoccum umdonicola and Lasiodiplodia pseudotheobromae (from Ecuador only) were also obtained. All isolates used in the pathogenicity trials produced lesions on inoculated plants, suggesting that the Botryosphaeriaceae contribute to the die‐back of S. parahyba trees. While the disease is clearly not caused by a single species of the Botryosphaeriaceae in either region, N. parvum has been introduced into at least one of the regions. This species has a broad host range and could have been introduced on other hosts.     

Phytophthora citrophthora,a new pathogen causing decline on horse chestnut in Turkey

Dieback symptoms were observed on horse chestnut trees planted approximately 40 years ago in Ankara, Turkey. Lesions at the stem bases of the affected trees were similar to those of ink disease on sweet chestnut. A Phytophthora sp. was isolated from the fine roots and soil samples collected around the stem bases by baiting using chestnut leaves. The pathogen was identified as Phytophthora citrophthora based on several morphological features and DNA sequences of the ITS region. Pathogenicity of P. citrophthora was tested by stem inoculation on 3‐year‐old horse chestnut saplings. P. citrophthora produced large cankers in 20 days and killed 40% of the saplings. This is the first report of P. citrophthora causing dieback on horse chestnut.     

Molecular diagnosis of Phytophthora lateralis in trees,water, and foliage baits using multiplex polymerase chain reaction

A polymerase chain reaction (PCR)‐based protocol for detection of Phytophthora lateralis in plant tissues and water is described. Base‐pair (bp) deletions in both of the ribosomal DNA internal transcribed spacer (ITS) regions in P. lateralis were used to design complementary PCR primer sequences that amplify a 738 bp fragment only if P. lateralis DNA is present in the sample. Universal control primers based on conserved sequences of the nuclear ribosomal small subunit are included in a multiplexed reaction, providing an internal check on the procedure. The universal primers amplify an approximately 550 bp fragment that is common to plants, protists, and true fungi. The procedure reliably detects P. lateralis in cedar stem tissues and in roots. Positive reactions were obtained with as few as 200 P. lateralis zoospores in water.     

Ceratocystis platani is killing plane trees in Istanbul (Turkey)

Ceratocystis platani was detected at several locations in the European side of Istanbul, causing severe dieback and mortality, mainly on Platanus × acerifolia imported from Italy approximately 160 years ago, but also on Platanus orientalis. In the work reported here, the causal agent of the dieback and mortality was identified based on morphological characteristics in culture and internal transcribed spacer (ITS) sequencing, confirming the presence of C. platani in Turkey. All analysed C. platani isolates from Istanbul shared the same ITS sequence with European C. platani strains available in the NCBI, but differed from other species previously ascribed to the Ceratocystis fimbriata species complex. Pathogenicity of C. platani was proven by inoculation onto 25‐cm‐long and 4–9 mm diam. P. orientalis twigs and the causal agent reisolated from symptomatic tissues. In a survey of 976 Platanus in streets and parks of Istanbul, 26.5% of trees showed symptoms of C. platani infections, whilst 5.6% were dead. The proportion of symptomatic trees was higher where management interventions, such as topping or pruning and felling infected trees had been carried out.     

Isolation of Phytophthora lateralis from Chamaecyparis foliage in Taiwan

Following the discovery in 2008 of Phytophthora lateralis in forest soil under old‐growth yellow cedar (Chamaecyparis obtusa var. formosana) in north‐east Taiwan, further sampling was undertaken in the same region. Soil, root and symptomatic foliage samples were collected from five separate sites where C. obtusa was the dominant species in cloud forests at ca. 1800–2500 m. Soil and fine root samples were baited with cedar needles; both direct isolation and cedar needle baiting were used on foliage samples. Phytophthora lateralis was obtained from soil at three of the sites, but only from three of the 27 soil samples overall. Only one of 25 root samples yielded the pathogen, and this was associated with infested soil. Three foliage samples with symptoms visible as dark brown to black frond tips also yielded P. lateralis; these came from two different sites. This is the first record of P. lateralis infecting the foliage of C. obtusa. Moreover, when some of the symptomatic Chamaecyparis foliage segments were incubated, sporangia of P. lateralis formed on the necrotic tissues, sometimes in the axils of needle segments. The study provides evidence that P. lateralis has both a soil/root infecting phase and an aerial or foliar infecting phase in Taiwan, which is consistent with its unusual combination of water‐dispersed (non‐papillate) and aerially dispersed (caducous) sporangia. It also demonstrates the importance of investigating the biology, aetiology and ecological behaviour of Phytophthoras in their native, endemic environments.