Characterisation of bacteria from poplars and willows exhibiting leaf spotting and stem cankering in New Zealand

During the unusually moist 1985–86 growing season, field grown poplars and willows exhibited on the current seasons growth, terminal and side shoot blackening, dieback, leaf spots and stem cankering. Bacteria isolated from infected tissues were: Pseudomonas syringae pv. syringae, P. fluorescens, Erwinia herbicola and Xanthomonas campestris pv. populi (poplar only). Isolates of P. syringae pv. syringae alone were pathogenic to the original hosts and evoked hypersensitive responses in tobacco leaves. The remaining bacteria were non-pathogenic and accordingly were considered to be components of the epiphytic microflora.     

Agrobacterium tumefaciens‐mediated transformation for targeted disruption and over expression of genes in the poplar pathogen Sphaerulina musiva

Sphaerulina musiva causes both leaf spots and cankers on poplar. Leaf spots can lead to defoliation and cankers on branches and primary stems can lead to stem breakage and tree mortality. The recent availability of both the S. musiva and Populus trichocarpa genomes offers a great opportunity to study host–pathogen interactions. To better understand the factors involved in S. musiva pathology, we present a strategy for the transformation of this species using Agrobacterium tumefaciens. Binary plasmids were generated with hygromycin B phosphotransferase (hph) flanked by upstream and downstream sequences of polyketide synthase‐like (PKS‐L1) gene to generate targeted gene disruptants by homologous recombination. Plasmids were also constructed for constitutive expression reporter genes eGFP and mCherry to help with histological characterization of the pathogen during infection. Gene knockouts were identified by PCR and confirmed by sequencing and Southern blotting. No significant differences were observed in melanin production between PKS‐L1 disruptants and wild type isolates. Colonies expressing reporter genes were identified by fluorescent stereomicroscopy. This method is a promising tool for the characterization of pathogen genes through reverse and forward genetics and for introducing markers for histopathological study.     

A medium to enhance identification of Septoria musiva from poplar cankers

A series of experiments was conducted to determine the relative tolerance in vitro of an isolate of Septoria musiva (a fungus that causes a severely damaging stem canker disease of poplars) for selected chemicals. Inhibition of diameter growth of this fungus on a V‐8 vegetable juice‐based medium with captan, chlorothalonil, iprodione, mancozeb and streptomycin sulphate at concentrations, respectively, of 50, 1, 10, 10, and 100 mg l^–1 was relatively low compared to inhibition of eight other fungi cultured from cankers on poplars. In addition, the presence of captan stimulated profuse sporulation of the fungus. These properties assisted in the identification of S. musiva from cankers resulting from artificial inoculation of poplar branches in the field.     

Effects of some bacteria (Pseudomonas spp. and Erwinia herbicola) on in vitro growth of Piptoporus betulinus

Summary Bacteria including Pseudomonas putida, Pseudomonas fluorescens biovar I, Pseudomonas fluorescens biovar V, Pseudomonas aureofaciens and Erwinia herbicola were isolated from discoloured zones in birch trunks. Antagonistic effects of these bacteria to growth of Piptoporus betulinus mycelium were tested in vitro, both in dual culture and using bacterial cell‐free culture filtrates. In dual cultures, P. putida was most effective at inhibiting mycelial growth of Piptoporus betulinus. Filtrates of P. putida inhibited growth of P. betulinus mycelium irrespective of filtrate concentration, incubation time of bacteria and timing of recording mycelium growth. The strongest antagonistic effect (inhibition of fungal growth) was observed on a medium containing 80% of sterile filtrate obtained from 15‐day‐old bacterial cultures. The highest stimulating effect on mycelium growth was noted on medium containing 80% filtrate obtained from 7‐day‐old E. herbicola cultures.     

欧美杨新型溃疡病病株与健株干部树皮中真、细菌优势种群分析

为探讨感染欧美杨新型溃疡病的中林46杨植株干部病斑中真、细菌优势种类,本研究对河南省濮阳市中林46杨健株、病株染病组织的可培养真菌、细菌优势种群进行了分析。结果表明:健康树皮样品分离真菌优势种群均为Alternaria alternata,染病株样品优势种群均为Fusarium solani,而且其菌株数量占所分离菌株总数的百分比均在85%以上;而在健康株3个处理中分离的细菌优势种群种类不同,仅Microbacterium在健株3个处理树皮样品中均有分布,但染病株3个处理树皮样品中的细菌优势种群均为Lonsdalea quercina,其菌株数量占所分离菌株总数的百分比均在67%以上。以上结果表明:中林46杨发病以后,F.solani和L.quercina分别变成了真菌和细菌的优势种群。     

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.     

Pathogenicity of fungi isolated from Quercus suber in Catalonia (NE Spain)

Summary Thirty‐four fungal species isolated from cork oak (Quercus suber) in Catalonia (NE Spain) during 1992–95 were tested for pathogenicity either in stem, leaf or root inoculations. Eleven species were found to be pathogenic on stem: Biscogniauxia mediterranea, Botryosphaeria stevensii, Diatrype cf. stigma, Endothia gyrosa, Fusarium solani, Graphium sp., Ophiostoma quercus, Phomopsis sp., Phytophthora cinnamomi, Sporendocladia bactrospora and an unidentified Coelomycete. Three fungi showed pathogenic effects on leaves: Dendrophoma myriadea, Lembosia quercina and Phomopsis quercella. No clear pathogenic effects were detected in the root inoculation experiment. Trunk pathogens were differentiated into two groups according to the effects induced in the inoculated plants; B. stevensii, Phomopsis sp. and P. cinnamomi caused the death of the inoculated plants and induced the formation of large cankers and vascular necroses. The other pathogenic species also produced severe cankers and vascular lesions, but no significant mortality was detected. Water stress increased the lesions caused by B. mediterranea and Phomopsis sp., but limited those of P. cinnamomi and the rest of the inoculated fungi. However, water stress did not significantly affect the damage caused by B. stevensii, which was the most virulent of the species tested. Leaf pathogens only showed their effects if the leaf cuticle was previously damaged. Lembosia quercina caused small dark lesions whereas D. myriadea and P. quercella produced large necrotic areas in well‐watered plants. The lesions caused by the last two fungi were reduced by water stress.     

Botryosphaeriaceae species causing canker and dieback of English walnut (Juglans regia) in Italy

English walnut (Juglans regia) is an important nut crop worldwide and is currently considered emerging in Italy. Botryosphaeriaceae fungi cause symptoms including cankers, discoloration and dieback, and several species are reported across the world on walnut. In this study, symptomatic trees from an orchard in Southern Italy showing branch dieback, cankers, wood discoloration and gummosis were surveyed. Three different fungi from the Botryosphaeriaceae were consistently isolated from symptomatic tissues. Representative isolates were characterized using morphological and molecular approaches based on conidial morphology, optimum growth temperature and the comparison of DNA sequence data from the ITS, tef1‐α and tub2 loci. Three species were identified: Botryosphaeria dothidea, Neofusicoccum mediterraneum and Neofusicoccum parvum. Pathogenicity tests on detached fruits and potted plants showed that all three species were pathogenic. To our knowledge, this is the first report of these Botryosphaeriaceae species causing canker and dieback on English walnut in Italy.     

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.     

Cultural and PCR‐based detection of Septoria musiva in inoculated hybrid poplar stems

The fungal pathogen Septoria musiva can be difficult to isolate from cankers that result from its colonization of poplar stems, and its persistence in these cankers has not been well studied. In order to compare cultural and polymerase chain reaction (PCR)‐based assays for detection of S. musiva in cankers, stems of susceptible hybrid poplar clone NC11505 were wounded and inoculated in August 2003. At 8, 16, 24 and 32 weeks after inoculation (October and December 2003, February and April 2004, respectively), 110 inoculated stems (plus controls) were harvested and a semiselective culture medium was used in attempts to detect the pathogen in bark and wood. Six chips of bark and six chips of underlying wood from one half of each canker were incubated on the semiselective medium for 2 weeks until pycnidia and conidia of S. musiva could be identified. The number of positive cankers and positive chips (out of six attempts per tissue per canker) was recorded. The remaining halves of cankers from subsets of 70 inoculated stems (plus controls) of those harvested in October 2003 and April 2004 were tested using a PCR‐based assay. Three chips of bark and three chips of underlying wood were ground, and DNA was extracted and then amplified using S. musiva‐specific primers designed from the internal transcribed spacer (ITS) region of nuclear rDNA repeats. The number of positive cankers and positive chips (out of three attempts per tissue per canker) was recorded. For both assays, the number of positive cankers and the number of positive chips per canker decreased with time. Using either assay, however, the pathogen was still detected from at least 49% of cankers at 32 weeks after inoculation.     

Sphaeropsis canker,a new disease of cypress in Greece

The fungus Sphaeropsis sp. is reported for first time in Greece to cause cankers on Cupressus sempervirens. The cultural characteristics on PDA, spore shape and size and canker morphology were identical to those of the fungus described as Diplodia pinea f. sp. cupressi in Israel. The cankers of Sphaeropsis sp. are characterized by resin exudation, with fissuring of the bark over a dry sector of the wood. The pathogenicity of the Sphaeropsis sp. was proved by artificial inoculations on selected cypress clones resistant and susceptible to Seiridium cardinale. The mean canker length of the susceptible clone was significantly more than that of the resistant clones. It appears that the defence reaction in cypress against both fungi is similar. In artificial inoculations on Pinus halepensis and Pinus pinea, Sphaeropsis sp. was nonpathogenic. During a 3‐year period, in an experimental plot in western Peloponnese, the fungus spread from one, initially, to 13 cypress clones. Drought stress during the summer appears to be the main factor predisposing the cypress plants to become susceptible to the fungus.     

Bark necrosis of Acer pseudoplatanus L. in northern Britain

Severe bark necrosis in large Acer pseudoplatanus was studied at a site in northern England. Bark death occurred late in the growing season or in the dormant season on two occasions and was preceded by a notably dry summer in both cases. Of several fungi isolated from cankers and areas of recent bark necrosis, Nectria coccinea and Diplodina acerina were demonstrated to be capable of causing limited necrotic extension of physical wounds in healthy A. pseudoplatanus. A third sp., Phomopsis pustulata, though the most frequently isolated fungus, showed no such ability. It is proposed that summer drought in association with a variety of weakly pathogenic fungi can induce local bark necrosis in A. pseudoplatanus.     

Relationships between Erwinia cancerogena Ur. and Aplanobacterium populi Ride

Detailed study of the bacteria Aplanobacterium populi and Erwinia cancerogena, employing cultural properties, disease symptoms, reisolations, serological properties, and filterable forms has shown close relationships between the two species. In both cases filterable forms, size 50–500 nm, exceptionally up to 900 nm were found. The filterable particles develop under the influence of a small spherical bacterial virus, size about 24 nm, which is adsorbed not only on vegetative cells but also on these filterable particles. The presence of bacterial virus and the formation of filterable particles influence directly the virulence of canker forming bacteria, notably the proliferation of tissues and the formation of tumour cankers.     

A new bacterial disease observed on Quercus myrsinifolia

This is the first report of a new bacterial disease occurring on Quercus myrsinifolia in the nurseries of the Kyushu district in Japan. The main symptoms are shoot and twig blight, which begins with black necrotic lesions. In addition, unclear blackish discolorations, bacterial ooze and water‐soaked lesions were observed as early symptoms, and spindle‐shaped cankers and stem cankers with irregular surface were observed as secondary symptoms. The blight symptoms observed on the current‐year shoots were first observed during the rainy season and continued until autumn (June–October). In summer, the symptoms were pronounced. A yellowish bacterium was consistently isolated from the affected shoots of Q. myrsinifolia, and on inoculation into healthy trees, it caused the same symptoms, which were more severe after wound inoculation than after non‐wound inoculation. Wound inoculation also caused the disease symptoms on Quercus spp., Fagus crenata and Lithocarpus glaber, but not on several other species of Fagaceae. The causal bacterium was identified as Xanthomonas arboricola based on its morphological, cultural and biochemical characteristics and genetic sequence analysis of 16S rDNA, 16S‐23S spacer regions and gyrB. To our knowledge, there are no previous records of xanthomonads causing shoot blight on fagaceous plants like Q. myrsinifolia. Therefore, we postulate that the shoot blight of Q. myrsinifolia is a new bacterial disease. We propose the name of the disease to be ‘bacterial shoot blight’ based on its main symptoms.     

Oak decline: Aerobic bacteria associated with declining Quercus cerris in Central Italy

Declining Turkey oaks (Quercus cerris) in Central Italy were studied. Bacteria were isolated from woody tissues between necrotic areas and healthy tissues beneath the bark, from apparently healthy tissues and from exudates oozing from the trunk in spring. A total of 98 bacterial isolates were collected. The determination of the bacteria was by fatty acid profiling and biochemical tests. Erwinia herbicola was frequently found both in necrotic and healthy tissues. Ice-nucleation active species were present. None of the species isolated was considered responsible for the decline     

Relationships between Oak powdery mildew incidence and severity and commensal fungi

Oak (Quercus robur) powdery mildew is a common and damaging fungal disease. In a local survey at Reading, UK, oak powdery mildew was common on trees of all height classes but was most common on trees of 3–9 m. A variety of other fungal species were commonly found growing in association with oak powdery mildew colonies. The abundance of such fungi was estimated through stratified sample surveys for 2.5 years. The taxa most commonly associated with oak powdery mildew were Acremonium sp., Trichoderma sp., Ampelomyces/Phoma sp. and Leptosphaerulina australis. Nearly 90% of mildew colonies were associated with L. australis, which is not generally considered as a mycoparasite or antagonist, in contrast with the other three fungi. Abundance varied between June and October surveys. Acremonium sp. abundance was greater in summer samplings, whereas L. australis and Trichoderma sp. abundances were greater in autumn samplings. Ampelomyces/Phoma sp. was never observed in the absence of powdery mildew. Relationships between the mildew‐associated fungi and oak powdery mildew appeared curved and differed significantly between sampling years. L. australis was positively correlated with the other three associated fungi studied when powdery mildew was also present . The variety and high population densities of the mildew‐associated fungi suggest that they may be important in determining the final density of oak mildew and the damage caused by it.     

Sphaceloma murrayae Jenk. & Grods., a pathogen new to Europe on Salix spp.

A Sphaceloma species causing greyish‐white leaf spots of several Salix species is described and illustrated. This is the first record for Europe of the mitosporic fungus identified as Sphaceloma murrayae, which was formerly known to occur only in New Zealand, Argentina and the US. In addition to information on symptoms and hosts, similar fungi occurring on willow, which can be confused with the new fungus are also discussed.     

Diplodia corticola and Phytophthora cinnamomi: the main pathogens involved in holm oak decline on Caprera Island (Italy)

Since 2008, severe and widespread tree decline and mortality has been observed at the main growing Quercus ilex L. (holm oak) forest on Caprera Island, Italy. To clarify the symptomatology and aetiology of this phenomenon, field surveys and isolations from symptomatic trees were carried out in summer 2010. Affected trees exhibited crown thinning, branch dieback, sunken cankers, epicormic shoots, exudates on branches and trunk, root losses and sudden death symptoms. Four fungal species belonging to Botryosphaeriaceae family, namely Botryosphaeria dothidea, Diplodia corticola, D. seriata and Neofusicoccum parvum, were isolated from cankers on trunk and branches, whereas three species of Phytophthora, namely P. cinnamomi, P. cryptogea and P. gonapodyides, were isolated from fine roots and rhizosphere soil samples. Isolates were identified using both morphological analysis and DNA‐based techniques. Pathogenicity trials on holm oak seedlings showed that all the isolated species are pathogenic. D. corticola proved to be the most aggressive species. Our results provide the first evidence for a combined involvement of D. corticola and P. cinnamomi in the aetiology of holm oak decline in Italy and suggest that these pathogens are not only important contributing factors in the onset of long‐term tree decline, but also may cause the rapid devastation of extensive oak ecosystems.     

First report of Sphaeropsis canker on cypress in South Africa

In this study a Sphaeropsis sp. is recorded as causing cankers on cypress in South Africa. These cankers are either found alone or on trees which are infected with the well-known cypress canker pathogen, Seiridium unicorne. When the two fungi occurred together, lesions could not be distinguished from each other, although the greater number of lesions were caused by the Sphaeropsis sp. The fungus appears to be identical to that described as Sphaeropsis sapinea f.sp. cupressi, in Israel, which is a fungus very different from the pine pathogen, Sphaeropsis sapinea. Pathogenicity tests showed that the Sphaeropsis sp. is significantly more pathogenic to Cupressus lusitanica than to Pinus roxburgii and Pinus elliottii. S. sapinea was pathogenic only to the Pinus spp. tested, and not to C. lusitanica. In contrast, the cypress pathogen Seiridium unicorne, was pathogenic to C. lusitanica and also to the two Pinus spp. tested.