Molecular phylogeny and pathotyping of Fusarium solani: a causal agent of Dalbergia sissoo decline

Sissoo (Dalbergia sissoo), commonly known as shisham, is amongst the finest woods of South Asia, but ‘wilt’ disease has caused a rapid decline in this species. The cause of the disease remains uncertain. The aim of this study is to identify the causal agent of the disease and characterize isolates made from diseased trees, based on genomic data and variations in virulence. Samples of infected roots, stems and the ooze exuded from infected trees were obtained from plants showing symptoms in different geographical regions of India for the isolation of microorganisms. Isolates were used to inoculate healthy plants. Based on the morphological characteristics, genus‐ and species‐specific PCR, and in silico analysis of 5.8S rDNA‐ITS regions, of the 38 fungal isolates, 24 and 14 were identified as Fusarium solani and Fusarium sp., respectively. In a pathotyping study, eighteen F. solani isolates, isolated from roots and stem parts of symptomatic plants, induced typical wilt symptoms when inoculated through soil and roots on D. sissoo seedlings of 1–15 months in age. The population of F. solani was the highest in infected roots and the lowest in parts of stems, gradually decreasing with height, and was isolated constantly up to approximately 40% height of the seedling. F. solani isolates used in inoculations were successfully re‐isolated from the rhizosphere, infected roots and wilted stems, as confirmed using isolate‐specific DNA fingerprints. Molecular phylogenies based on rDNA‐ITS sequences showed that the 38 isolates fell into 2 groups. Group I comprised of F. solani isolates from D. sissoo and F. solani sequences in the NCBI GenBank database, whereas group II included Fusarium isolates other than F. solani. These results are helpful in developing integrated control measures for this highly variable pathogen and to establish a base for future population studies.     

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.     

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.     

Incidence of dieback disease following fungal inoculations of sexually and asexually propagated shisham (Dalbergia sissoo)

Shisham (Dalbergia sissoo) is an important multipurpose tree with great economic importance, but this tree has been devastated by dieback disease. Seedlings and asexually propagated (cuttings) plants were artificially inoculated with four fungi (Fusarium solani, Botryodiplodia theobromae, Curvularia lunata and Ganoderma lucidum) to evaluate the potential role of these fungi in shisham dieback disease. Results at 2 years revealed that highest disease was caused by inoculation of F. solani (31.39%), followed by B. theobromae (19.042%) and C. lunata (12.22%), but no dieback disease was caused by G. lucidum. During both years, seedlings exhibited greater susceptibility to disease (17.24%) compared to cuttings (7.83%). In particular, F. solani caused more disease in seedlings (46.18%) compared to cuttings (16.61%). With the F. solani inoculations, maximum disease rate was observed at 8 weeks post‐inoculation both in seedlings (77%) and in cuttings (31%), but the maximum disease increase was observed at 4–5 weeks post‐inoculation. Analysis of variance showed significant differences among the different fungi and also between seedlings and cuttings. F. solani can be considered as a major fungal pathogen contributing to dieback disease of shisham, and asexual propagation can reduce the severity of dieback.     

Bacillus pumilus and Stenotrophomonas maltophilia as two potentially causative agents involved in Persian oak decline in Zagros forests (Iran)

The oak decline is known as one of the most destructive complex diseases causing high economic losses around the world, especially in Iran. The main objective of the present study was to investigate the possible role of bacteria as causative agents of oak decline in the Zagros forests of Iran. To do this, stem, root and leaf samples were taken from symptomatic Persian oak trees (Quercus brantii) in different zones of Zagros forests (Ilam Province, Iran). From 150 bacterial isolates, 20 showed pathogenicity against Geranium seedlings. Among 20 hypersensitivity test positive strains, four strains showed pathogenicity against oak saplings. Based on morphological and 16S rRNA gene sequence analysis, three strains were identified as Bacillus pumilus and one strain as non‐sporulating Gram‐negative Stenotrophomonas maltophilia. Pathogenicity studies of different B. pumilus and S. maltophilia strains revealed that they have potential to cause the disease in oak saplings and symptoms of disorder in Persian oak trees. To our knowledge, there are no previous records of B. pumilus and S. maltophilia causing decline on Fagaceous trees like Q. brantii. More detailed field and molecular studies are required to confirm the absolute role of such bacteria in occurrence of oak decline in Zagros forests.     

First report of Sydowia polyspora causing disease on Pinus pinea shoots

The fungus Sydowia polyspora is frequently isolated from conifers worldwide and is considered a pathogen on several hosts. Stone pine (Pinus pinea) is one of the most important forestry species throughout the Mediterranean basin due to the value of the edible pine nut. Stone pines showing tip dieback, needles with tan‐ to yellow‐coloured lesions and shoot death, observed in stands in Portugal, were sampled for analysis. Fungal colonies covered with cream‐coloured spore masses, were consistently obtained. Morphological and phylogenetic analyses of the ITS rDNA region enabled identification of these isolates as S. polyspora. Inoculation tests showed that the fungus caused lesions on excised P. pinea shoots. The symptoms observed might have a negative effect on pine nut production, and thus, evaluation of the impact of this disease is of relevance to future research. This paper is the first to report S. polyspora causing disease on P. pinea.     

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.     

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.     

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.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

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.     

Infection approach of Diplodia sapinea,the causal agent of tip blight of Pinus tabulaeformis in China

To clarify the infection approach of Diplodia sapinea, a pathogen that causes tip blight of Pinus tabulaeformis, the infection process of the pathogen in needles was observed using scanning electron microscopy (SEM). In addition, the disease incidence on branches damaged by Aphrophora flavipes (Hemiptera: Cercopidae) and Dioryctria splendidella (Lepidoptera: Pyralidae) in the forest was also investigated. Then, branches and needles of P. tabulaeformis were inoculated using the D. sapinea spore suspension under indoor and field conditions. The results showed that the damage caused by A. flavipes could aggravate the occurrence of tip blight of P. tabulaeformis to some extent. Moreover, the pathogen could also penetrate 1‐, 2‐ and 3‐year‐old pine needles through stomata in the field. The pathogen infected the 1‐year‐old branches first and then gradually spread to 2‐ and 3‐year‐old branches.     

Providencia rettgeri as the causal agent of the brown slime flux of Populus tomentosa

Brown slime flux seeps slowly out of wounds and flows down the bark of roadside Populus tomentosa in Beijing, China. Two bacterial isolates, SL2‐2 and SL3‐3, obtained from the brown slime flux were identified as Providencia rettgeri based on 16S rRNA gene sequencing. After inoculation into the bark of roadside P. tomentosa with sterile deionized water as a control, both SL2‐2 and SL3‐3 triggered the seepage of brown slime flux from the wounds; no slime flux arose from the control. In conclusion, P. rettgeri was the pathogenic bacterium causing brown slime flux on P. tomentosa probably by inhibiting wound healing. To our knowledge, this is the first report of P. rettgeri as a pathogen of plants.     

Brenneria spp. and Rahnella victoriana associated with acute oak decline symptoms on oak and hornbeam in Iran

Decline diseases of forest trees are complex syndromes not attributable to single causal factors. In Iran, symptoms of decline disease have been observed in a number of native forest species including Quercus castaneifolia (chestnut‐leaved oak), Q. brantii (Persian oak) and Carpinus betulus (hornbeam). The symptoms are prevalent in the northern forests and the Zagros mountain forests. There are parallels between the disease in Iran and acute oak decline (AOD) reported in the UK, specifically the presence of weeping cankers, which have been attributed to a polybacterial complex wherein Brenneria goodwinii is considered a key necrogen. Based on the AOD symptomatology, and as a first step towards discovering potential causal agents of the stem weeping symptoms of affected trees in Iran, necrotic tissues were tested primarily for the presence of B. goodwinii. Symptomatic Q. castaneifolia and C. betulus from the Mazandaran Province and symptomatic Q. brantii from Kohgiluyeh and Boyerahmad Province were sampled. Isolation and culture on a selective medium yielded uniform bacterial colonies. Isolates were characterized using phenotypic and genotypic (DNA sequencing) tests. The isolates were phenotypically identical to members of Pectobacteriaceae and Yersiniaceae, specifically Brenneria and Rahnella spp. The nucleotide sequences of the 16S rRNA and housekeeping genes gyrB, infB and atpD (MLSA) amplified via PCR demonstrated that the isolates from the trees in Iran were indeed B. goodwinii, B. roseae subsp. roseae, Rahnella victoriana and an unknown species of Brenneria. Most bacteria isolated from non‐symptomatic trees were Gram‐positive, and Pseudomonas spp. were dominant, but bacterial species isolated from the diseased trees were not detected in healthy trees. Hypersensitivity response tests were positive, but inoculation on saplings was more variable with internal necrosis developing only once in the test period. Therefore, further testing is required. This is the first report of the incidence of B. goodwinii, B. roseae subsp. roseae, R. victoriana and Brenneria sp. associated with acute oak decline‐like symptoms on Q. castaneifolia, Q. brantii and C. betulus across the western forests of Iran and in the world.     

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.     

Genetic diversity of pine‐parasitic nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus in China

The internal transcribed spacer (ITS) regions of rDNA have been routinely employed for identification and phylogenetic analysis of many nematode species. In this study, the intra‐ and interspecies ITS genetic diversity of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus was evaluated. Ninety‐one isolates of the two nematode species collected from 14 Chinese provinces, Japan and Korea were used for ITS‐PCR and sequencing. An unweighted pair group cluster analysis dendrogram clustered them as two B. mucronatus and one B. xylophilus independent clades. Principal component analysis showed the phylogenetic relationship of the two nematode species more clearly; B. mucronatus isolates were separated into more than four groups, whereas B. xylophilus isolates still clustered into a group. The results of the Mantel test indicated the correlation of genetic distance matrices and geographic distance matrices was significant for both nematode species. The genetic differentiation coefficient (G_st) and gene flow (N_m) of B. mucronatus were 0.341 and 1.091, respectively, suggesting the importance of landscape heterogeneity and considerable obstacles for genetic exchange among B. mucronatus isolates in China. However, G_st and N_m of B. xylophilus were 0.188 and 2.151, respectively, very different compared to B. mucronatus. This could be owing to the short‐term introduction of B. xylophilus into China and a rapid spread through anthropogenic pathways. Our work adds to the understanding of the genetic diversity and genetic relationship of the two pine‐parasitic nematode species, and will aid in controlling them in the future.     

The first record of Botryodiplodia canker in Poland

This study describes the first observation of Botryodiplodia canker in the Western Carpathians in south‐eastern Poland caused by Botryodiplodia hypodermia (Sacc.) Petr. (syn. Sphaeropsis hypodermia, S. ulmicola). The canker occurred on an approximately 17‐year‐old Ulmus glabra sapling in a mixed conifer/deciduous stand with elm trees severely damaged by Dutch elm disease. This paper describes disease symptoms and provides information on the macro‐ and micromorphology of the fungus isolated from the cankered tissues. The results of BLAST search using DNA sequences obtained for our cultures and subsequent phylogenetic positioning of the fungus among closely related Botryosphaeriaceae indicate that the species is much more closely related to Phaeobotryon than to the other Botryodiplodia or Sphaeropsis species. Moreover, a total of 16 polymorphisms within the ITS region were detected between S. ulmicola associated with Botryodiplodia canker in North America and B. hypodermia associated with the canker observed in Poland. Thus, the “European” variant of “Sphaeropsis” ulmicola can now be easily identified with our barcode sequences. The Botryodiplodia canker is much less prevalent in Europe than in North America. Differences in virulence of “American” and “European” linages and differences in susceptibility of various elm species may be the reason for the higher prevalence of the disease in North America.     

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD), which is a major problem in East Asia and West Europe. Quick identification of PWN is needed to prevent the dispersal of PWD to healthy forests. Various detection methods of PWN have been developed using anatomical characters and molecular markers. These methods are not suitable for rapid diagnosis because it is difficult to distinguish B. xylophilus from the non‐pathogenic species Bursaphelenchus mucronatus based on morphological characters without expertise in nematode taxonomy and most PCR or isothermal amplification detection methods require time‐consuming processes. In this study, we developed an on‐site PWN detection method using a recombinase polymerase amplification (RPA) assay with a novel extraction buffer (DAP buffer). This new PWN detection method is able to extract genomic DNA from PWN in pinewood by simple buffer consisting of sodium hydrate, polyethylene glycol 200 and dimethyl sulfoxide in 10 min without using the experimental devices and able to distinguish between B. xylophilus and other Bursaphelenchus spp. by amplifying the species‐specific 5S rDNA fragment of B. xylophilus in 10 min. Taken together, our protocol can obtain the result for the detection of PWN in pine tree samples within 30 min. This result suggests that RPA/DAP assay is much faster, easier and cheaper than the conventional methods for detecting PWN.