Identification and pathogenicity of Alternaria alternata causing leaf spot and blight disease of Ailanthus excelsa in India

Alternaria leaf spot of Ailanthus excelsa is generally considered as minor disease in India. Recently, severe disease outbreaks were recorded in the nursery of the Forest Research Institute, Dehradun, progeny trial at Jhumpa, Haryana, and in a nearby farm field. Leaf symptoms included small circular, brown, necrotic spots with a chlorotic halo. With severe infections, leaf spots coalesced and resulted in leaf blight. A small‐spored Alternaria with concatenated conidia was isolated consistently from the leaf samples with spot symptoms. Sequence analyses of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and the translation elongation factor 1‐alpha (tef‐1α) gene region of two fungal isolates confirmed the species as A. alternata. In detached leaf assays, both isolates produced symptoms similar to those observed on the nursery/field‐grown plants. To validate the detached leaf assay result, pathogenicity was also demonstrated on whole plants in a glasshouse. Koch's postulates were fulfilled by re‐isolating A. alternata from the inoculated leaves. This work is the first to confirm that A. alternata is associated with leaf spot and blight disease of A. excelsa in India.     

Biocontrol potential of Pseudomonas azotoformans,Serratia marcescens and Trichoderma virens against Fusarium wilt of Dalbergia sissoo

Wilt disease caused by Fusarium solani is a serious constraint to Dalbergia sissoo (shisham) plantations in northern India. In this study, the antagonistic potential of 40 bacterial isolates recovered from rhizophere soil of healthy shisham trees, and a well‐characterized Trichoderma species (Trichoderma virens) were tested for their possibility as biocontrol agents for F. solani. Two promising isolates (S1 and S15) were identified which inhibited pathogen growth, caused chitin degradation, produced siderophores and solubilized phosphate in vitro. Isolate S15 scored highest for hydrogen cyanide (HCN) production while isolate S1 was a non‐HCN producer. These two isolates were identified as Serratia marcescens (S1) and Pseudomonas azotoformans (S15) following sequence analysis of 16S rDNA. In dual culture assays, T. virens caused 80% inhibition of mycelial growth of the test fungus. The three selected antagonists when tested in planta in the glasshouse completely suppressed production of wilt symptoms on 12‐month‐old shisham plants. Further work is needed to ascertain the potential of these isolates to be used as biocontrol agents to manage shisham wilt under field conditions.     

Wood‐rotting basidiomycetes are a minor component of fungal communities associated with Acacia hybrid trees grown for sawlogs in South Vietnam

Acacia hybrid (Acacia mangium × Acacia auriculiformis) clones are widely planted in Vietnam with a total of approximately 400,000 ha to meet the demand for pulpwood, sawn timber and wood chip exports. Silvicultural techniques such as pruning and thinning have been applied to improve productivity and sawlog quality of Acacia hybrid plantations. However, those techniques may also create opportunities for wood decay fungi to enter the Acacia hybrid stems through wounds and cause stem defects that reduce sawlog quality and the value of the plantation. The presence of fungal decay agents in Acacia hybrid trees was examined in two Vietnamese plantations. In July 2011, just prior to a second thinning, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Phan Truong Hai for the isolation of fungi. In July 2012, approximately 18 months after pruning and thinning treatments, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Nghia Trung for the isolation of fungi. DNA sequencing of the rDNA ITS identified the isolates. In May 2015, approximately 4 years after thinning and fertilizer treatments, discoloured and decayed wood samples were taken from the above (7‐year‐old) Acacia hybrid plantation at Phan Truong Hai for fungal identification. DNA was extracted directly from discoloured and decayed wood samples and fungal rDNA ITS amplicons sequenced on a Roche 454 sequencer. The results showed that silvicultural treatments did not affect the fungal communities associated with discoloured and decayed wood of Acacia hybrid plantation at Phan Truong Hai. A total of 135 fungal species or OTUs (operational taxonomic units) were identified, including 82 members of Ascomycota and 52 Basidiomycota.     

Diseases of eucalypts in the central and northern provinces of Mozambique

In the mid-1970s, Mozambique embarked on several forestry projects to promote and establish large plantation areas with eucalypt trees. The planted species included Corymbia citriodora, Eucalyptus camaldulensis, E. saligna and E. tereticornis. Similar to other regions of the world, pests and pathogens pose a significant threat to eucalypt plantations in Mozambique, but little is known regarding the diseases of trees in the country. The aim of this study was to provide the first detailed consideration of the possible disease problems affecting plantation- grown eucalypt species in the central and northern provinces of Mozambique. Isolates of fungal pathogens were obtained from material displaying disease symptoms collected during two field surveys conducted in 2010 and 2011. Representative fungal isolates were identified using DNA sequence data. Stem canker diseases caused by fungi in the Botryosphaeriaceae, Cryphonectriaceae and Teratosphaeriaceae, as well as leaf spots caused by species of Calonectria, Pilidiella, Pilidium and Phakopsora myrtacearum were commonly encountered. Their relative importance to the future of planation forestry in Mozambique is discussed.     

Ophiostomatoid fungi isolated from three different pine species in Argentinian Patagonia

Pine plantations in Argentinian Patagonia cover ca. 95,000 ha in Chubut, Río Negro and Neuquén provinces. Exotic bark beetles (Orthotomicus laricis, Hylastes ater and Hylurgus ligniperda) commonly occur in freshly cut logs, stumps and slash. These beetles are vectors of “ophiostomatoid” fungi which include primary tree pathogens as well as important agents of blue stain. The aim of this study was to identify these beetle‐associated fungi. Sawing mills and pine plantations were surveyed three consecutive years. Fungal isolates from stained logs, processed wood and insect galleries were identified based on morphological and DNA sequence comparisons of ITS and β‐tubulin gene regions. Two Grosmannia, one Graphilbum and three Ophiostoma species were identified. Ophiostoma piliferum and O. peregrinum sp. nov. were the most frequently isolated taxa. O. peregrinum occurred in all provinces, colonizing different conifer species and, interestingly, also the native broadleaved species Nothofagus dombeyi. Pine plantation forestry in southern South America includes Argentina, Brazil, Chile, Paraguay and Uruguay. Emerging data from Argentina, Chile and Uruguay revealed some coincidences between these countries, but also several differences, probably, as a result of multiple introduction events.     

An agglutination test for fast and specific detection of Erwinia psidii

Dieback and wilt, caused by Erwinia psidii (Ep), is one of the most important emergent diseases of Eucalyptus spp. in Brazil. Currently, pathogen detection relies on isolation of bacteria from infected plant tissue and either identification based on morphological, physiological and biochemical tests or DNA amplification using the polymerase chain reaction (PCR), which in many cases is laborious and cumbersome. Considering the need for a simpler and more rapid, yet reliable, method for detecting the pathogen, we obtained a polyclonal antibody (anti‐Ep) and developed an agglutination test for specific detection of E. psidii. The antiserum was produced against the E. psidii strain LPF534 and tested against 101 E. psidii isolates from Eucalyptus spp.; three E. psidii isolates from Psidium guajava; 23 Ralstonia solanacearum and 18 Xanthomonas axonopodis isolates pathogenic to Eucalyptus spp.; and seven endophytic isolates from Eucalyptus spp., three of which are phylogenetically related to the genus Erwinia. Results of direct ELISA indicated that a concentration as low as 3.5 µg/ml of the anti‐Ep antibody was able to detect the E. psidii antigen and that the antibody did not cross‐reacted with other bacteria pathogenic and non‐pathogenic to Eucalyptus spp. In the agglutination test, the anti‐Ep antibody showed positive reaction with all strains of E. psidii tested whereas cross‐reaction with none of the strains that belong to other taxonomic groups was observed. The agglutination test showed a detection limit of 10⁵ colony‐forming units (CFU)/ml, and its specificity was the same as that obtained by PCR amplification using E. psidii‐specific primers. These results demonstrate that the agglutination test developed here is a useful tool for specific, fast and inexpensive detection of E. psidii although only operational on pure bacterial suspensions and not yet directly from infected tissues.     

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.     

Mating type ratios and pathogenicity in Diplodia shoot blight fungi populations: Comparative analysis

Diplodia sapinea and Diplodia scrobiculata are opportunistic pathogens of Pinus species. Several studies about taxonomy, impact and epidemiology of these fungi have been conducted in previous years, which have provided useful information and have raised new issues. These diseases produce a considerable impact on plantations resulting in significant economic losses. The main aims of this study are to increase the knowledge of the potential of genetic exchange and the relative aggressiveness of these organisms that can persist in healthy tissues of asymptomatic trees. A collection of 250 isolates among which are 149 strains collected from Pinus radiata plantations in Basque Country (Spain) and 101 strains from different countries was included in this work. Mating type ratios were analysed and compared using the structure of the MAT locus (MAT1‐1‐1 and MAT1‐2‐1). Inoculations of Pinus radiata seedlings were performed in a biosafety greenhouse (P2) to confirm pathogenicity of isolates and compare their aggressiveness. The frequency of occurrence of both idiomorphs of D. sapinea in Basque Country isolates was close to 1:1, however, for collection of isolates of this fungus from around the world, the ratio was 1:2. Furthermore, the spatial distribution of the two mating types in the Basque Country was random. Despite no detection of a sexual state, these results could suggest sexual reproduction behaviour. The pathogenicity of all strains in the collection was confirmed. Although aggressiveness (in terms of lesion lengths resulting from inoculation) varied greatly, no statistically significant effects of MAT type or pathogen species were detected.     

Phosphorus,nitrogen and potassium nutrition on Calonectria leaf blight in eucalypt plants

Calonectria pteridis causes Calonectria leaf blight (CLB), and consequently severe defoliation in eucalypt plantations, which results in losses in wood volume. To reduce the negative impacts of this disease in eucalypt, this study aimed to assess the application of different doses and combinations of the macronutrients N, P and K on the percentage of symptomatic leaf area (SLA) and defoliation induced by the pathogen. Cuttings of a clone of Eucalyptus grandis were transplanted to pots containing soil that received different dose combinations of N, P and K, according to an incomplete factorial design. At 200 days after transplanting, leaf samples were analysed for N, P and K contents, and then, the plants were inoculated. Forty‐five days post‐inoculation, SLA and percentage of defoliation were quantified. Potassium doses above 75 mg/dm³ of soil significantly reduced SLA and defoliation. The influence of N and P on defoliation was dependent on K doses, but both reduced symptomatic leaf area. The best control of the disease, expressed by decreased defoliation and symptomatic leaf area, was achieved with leaf content of N, P and K of 9.8, 0.8 and 10.4 g per kg leaf, respectively, obtained with doses of 55, 82.5 and 143 mg/dm³of soil, respectively. Therefore, N, P and K nutrition can be a component of an integrated management programme for the control of CLB in eucalypts.     

Outbreak of shoot blight and dieback of Eucalyptus spp., caused by Pseudoplagiostoma eucalypti in Brazil

An outbreak of a new and severe disease was observed in Eucalyptus plantations of Bahia state, Brazil. An Ascomycota fungus has been frequently associated with the main symptoms of the disease namely leaf spot, branch cankers, shoot blight, defoliation, and dieback. Based on morphological characteristics, phylogenetic analysis (ITS and TEF-1α genes), and pathogenicity test on Eucalyptus plants, Pseudoplagiostoma eucalypti was identified as the causal agent of the disease. Although P. eucalytpi has been known from in Brazil since 1998, this is the first report of it causing severe disease and die-back on Eucalyptus spp. and we also record new symptoms associated with the pathogen.     

The emergence of beech leaf disease in Ohio: Probing the plant microbiome in search of the cause

American beech (Fagus grandifolia) is the target of a newly emerging disease in North America called beech leaf disease (BLD) that affects and disfigures leaves and which can lead to tree mortality. Beech leaf disease may be caused by a newly recognized subspecies of the anguinid nematode Litylenchus crenatae subsp. mccannii, but the associations of this nematode with bacterial and fungal taxa are unknown. We examined microbial communities associated with beech leaves affected by BLD in a 16‐year‐old American beech plantation using molecular methods. We detected L. crenatae subsp. mccannii in anywhere from 45% to 90% of leaves depending on the degree of visual BLD symptoms. Approximately 37% of asymptomatic leaves contained L. crenatae subsp. mccannii, whereas 90% of buds associated with symptomatic leaves contained L. crenatae subsp. mccannii. We found that fungal communities on leaves and buds were unaffected by BLD, but bud and leaves had significantly different fungal communities. Bacterial communities on buds also were unaffected by BLD, but bacterial communities were significantly different between symptomatic and asymptomatic leaves suggesting that the nematode could be altering the community of bacteria on the leaves. Clone libraries indicate that Wolbachia, an intracellular endosymbiont of arthropods, was found only on symptomatic leaves and buds associated with either symptomatic or asymptomatic leaves. In addition, only symptomatic leaves contained taxa in the genus Mucilaginibacter, which previous studies suggest could produce exopolysaccharides. These bacterial taxa could represent a marker for the vector of L. crenatae subsp. mccannii that enables spread between trees and a possible endosymbiont that could facilitate nematode feeding and establishment on nematode infested leaves. Our results are the first to examine changes to the leaf microbiome of this newly emerging pest and may aid identification of mechanisms associated with the spread and success of L. crenatae subsp. mccanni.     

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.     

Decline of Paulownia tomentosa caused by Trametes hirsuta in Serbia

During the monitoring of the health status of nurseries and plantations in Serbia, a decline in a 5‐year‐old Paulownia tomentosa plantation was recorded. Trees displayed symptoms of dieback, massive breaking at different positions, the appearance of decay and fungal fruitbodies at the stems. Using standard isolation methods, white colonies with cottony surfaces and regular growth were obtained from the decaying wood samples, and after the oxidation degree analysis, it was determined that these isolates belonged to Davidson's group 6, indicating white rot basidiomycetes. To identify the isolated fungus, the ITS region of one selected isolate was sequenced. Based on the morphological analysis of the obtained colonies, collected fruitbodies and ITS sequence analysis, this fungus was identified as Trametes hirsuta. This is the first report of T. hirsuta on Paulownia tomentosa in Serbia. Irregularly performed pruning as the possible infection route and the implications of these findings are discussed.     

Outbreak of powdery mildew caused by Sawadaea negundinis on boxelder in Iran

Boxelder (Acer negundo) is planted as ornamental and shade tree in many countries including Iran. During July 2016, boxelder trees in parks and streets of different localities in East Azarbaijan province, Iran were found to be heavily infected with powdery mildew. Whitish powdery fungal mass developed mainly on the lower leaf surface. The infected young leaves became bubbled, curled and crinkled, giving affected plants an unpleasant appearance. During September, scattered, dark brown, spherical chasmothecia were produced on the lower leaf surface. The causal agent was identified as Sawadaea negundinis based on a combination of morphological characteristics of sexual and asexual morphs and sequence data of the rDNA internal transcribed spacer region (ITS1‐5.8S‐ITS2). This study provides the first report on the occurrence of S. negundinis on boxelder in Iran.     

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.     

Bacterial shoot blight of sweet crab apple caused by Pseudomonas viridiflava

Sweet crab apple (Malus coronaria) is a popular ornamental tree planted in gardens and urban forests in South Korea. In 2018, severe symptoms were observed on street plantings of sweet crab apple in Jinju, South Korea including shoot blight: shoot tips, twigs and foliage of infected trees turned brown, and dried completely. The causal agent isolated from the disease lesions was identified as Pseudomonas viridiflava based on biochemical tests, pathogenicity, and 16S rRNA and rpoD gene sequencing. On artificial inoculation, this pathogen not only caused shoot blight on sweet crab apple, but also bulb rot and leaf blight on onion. Here, we report the occurrence of bacterial shoot blight caused by P. viridiflava in sweet crab apple for the first time in the world.     

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.     

First report of Fusarium solani causing wilt of Melia dubia

Melia dubia, a multipurpose tree species, is gaining importance to meet the demand supply gap of timber, plywood and pulpwood . In June 2016, a serious outbreak of wilt disease was observed in M. dubia seedlings planted in the Central Nursery of Forest Research Institute (FRI), Dehradun, India. The disease led to the destruction of one hundred thousand (100,000) seedlings. Earlier in June 2012, serious wilting of M. dubia seedlings was observed in Haryana, India. The pathogen was identified as Fusarium solani following standard laboratory procedures and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). The pathogenicity of three isolates has been proved under greenhouse conditions. This is the first report of F. solani causing wilt of M. dubia.     

Studies on leaf spot and shoot blight of Eucalyptus caused by Cylindrocladium quinqueseptatum

In glasshouse experiments, Cylindrocladium quinqueseptatum, which was associated with outbreaks of leaf spot and shoot blight in a young plantation of Eucalpytus microcorys in north Queensland during the 1982 and 1983 autumns, was pathogenic on seedlings of E. microcorys and caused varying degrees of leaf spot and shoot blight in ten species of Eucalyptus from major taxonomic groupings of the genus, When the outbreaks occurred, the weather was unusually showery with minimum temperatures above 16°C. In vitro studies showed that free water was essential for conidial germination, which occurred between 14–32°C. The optimum for mycelial growth was 28.5°C. In vivo studies showed that leaf penetration. by germinated conidia was via the stomata. Mancozeb was more effective at controlling the disease than benomyl, captan or copper oxychloride. The potential importance of C. quinqueseptatum and options for control in young Eucalyptus plantations are discussed.