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.     

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.     

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.     

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.     

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.     

Pseudomonas associated with Bursaphelenchus xylophilus,its insect vector and the host tree: A role in pine wilt disease?

In this study, we characterized the diversity of Pseudomonas associated with Bursaphelenchus xylophilus, its insect vector (Monochamus galloprovincialis) and its host (Pinus pinaster), by a culture‐independent approach using rpoD clone libraries. Clone libraries of Pseudomonas rpoD were obtained from B. xylophilus, M. galloprovincialis and infected P. pinaster. Most M. galloprovincialis and B. xylophilus sequences grouped together in the P. fluorescens group. Genes related to xenobiotics degradation and phenylacetate synthesis were present in the genomes of the type strains closely related to sequences retrieved from the nematode libraries. Results demonstrated that the nematode, during its life stages inside the tree, maintains a diverse Pseudomonas community that is closely related to the one associated with the insect vector. These bacteria might contribute to degradation of xenobiotics and tree weakening during the nematode tree infection.     

Control of Hymenoscyphus fraxineus,the causal agent of ash dieback,using composting

Viability of Hymenoscyphus fraxineus inocula following temperature treatments for different exposure times was examined in vitro and in aerated flask‐ and large‐scale composting tests using green waste. After an exposure for up to 10 days at 20°C, 97.3% of H. fraxineus mycelium and pseudosclerotia plate cultures remained viable. No viability was detected following a 3‐day exposure to 40°C or a 1‐day exposure to 45°C although pseudosclerotia were more tolerant than mycelium to an exposure to 35°C. Primordial apothecia of H. fraxineus emerged from 62%–100% of infected ash rachises collected from two infected sites and stored at 4°C for 0–5 months; exposure to compost for up to 10 days at 20°C did not affect this emergence. No emergence of H. fraxineus apothecia was observed from ash rachises that were exposed to compost at 45°C for 1 day or at 35°C or 40°C for 3 days in flasks or at 40°C for 1 day or at 30°C for 5 days in a large‐scale composting system. Based on a fitted model, estimates of the survival of H. fraxineus inoculum in infected ash rachises exposed to compost at 50°C for 1 day were 0.081% of that in the untreated H. fraxineus ash rachis inoculum. Increasing loss in viability of H. fraxineus inoculum in infected ash rachises during longer and warmer exposures to compost at 35°C–45°C corresponded with a reduced concentration of pathogen DNA detected in the rachises using real‐time PCR. However, exposure of rachises to compost at >53°C resulted in a smaller reduction in pathogen DNA detected than exposure to compost at lower temperatures, possibly due to the inhibition of enzymatic degradation of DNA at elevated temperatures.     

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

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

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.     

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.     

Dothistroma spp. in Western Ukraine and Georgia

Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.     

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.     

Genetic diversity of the myrtle rust pathogen (Austropuccinia psidii) in the Americas and Hawaii: Global implications for invasive threat assessments

Since the myrtle rust pathogen (Austropuccinia psidii) was first reported (as Puccinia psidii) in Brazil on guava (Psidium guajava) in 1884, it has been found infecting diverse myrtaceous species. Because A. psidii has recently spread rapidly worldwide with an extensive host range, genetic and genotypic diversities were evaluated within and among A. psidii populations in its putative native range and other areas of myrtle rust emergence in the Americas and Hawaii. Microsatellite markers revealed several unique multilocus genotypes (MLGs), which grouped isolates into nine distinct genetic clusters [C1–C9 comprising C1: from diverse hosts from Costa Rica, Jamaica, Mexico, Puerto Rico, and USA‐Hawaii, and USA‐California; C2: from eucalypts (Eucalyptus spp.) in Brazil/Uruguay and rose apple (Syzygium jambos) in Brazil; C3: from eucalypts in Brazil; C4: from diverse hosts in USA‐Florida; C5: from Java plum (Syzygium cumini) in Brazil; C6: from guava and Brazilian guava (Psidium guineense) in Brazil; C7: from pitanga (Eugenia uniflora) in Brazil; C8: from allspice (Pimenta dioica) in Jamaica and sweet flower (Myrrhinium atropurpureum) in Uruguay; C9: from jabuticaba (Myrciaria cauliflora) in Brazil]. The C1 cluster, which included a single MLG infecting diverse host in many geographic regions, and the closely related C4 cluster are considered as a “Pandemic biotype,” associated with myrtle rust emergence in Central America, the Caribbean, USA‐Florida, USA‐Hawaii, Australia, China‐Hainan, New Caledonia, Indonesia and Colombia. Based on 19 bioclimatic variables and documented occurrences of A. psidii contrasted with reduced sets of specific genetic clusters (subnetworks, considered as biotypes), maximum entropy bioclimatic modelling was used to predict geographic locations with suitable climate for A. psidii which are at risk from invasion. The genetic diversity of A. psidii throughout the Americas and Hawaii demonstrates the importance of recognizing biotypes when assessing the invasive threats posed by A. psidii around the globe.     

“Candidatus Phytoplasma solani” associated with Eucalyptus witches’ broom in Iran

During summer of 2015, Eucalyptus camaldulensis plants showing witches’ broom, little leaf and general yellowing of the foliage were observed in west of Fars and Khozestan province of Iran. DNA from samples of 22 symptomatic and two asymptomatic trees was extracted and subjected to molecular analyses. Nested‐PCR test using R16F2n/R16R2 primers confirmed phytoplasma presence in 63% of symptomatic Eucalyptus plants. Sequence analysis along with virtual RFLP of the 16S ribosomal DNA allowed to classify three Eucalyptus witches’ broom strains into the “stolbur” (“Candidatus phytoplasma solani”) 16SrXII‐A subgroup. Comparison of the secA and secY gene sequences with sequences deposited in GenBank confirmed the phytoplasma identity. Real and virtual RFLPs of the amplified secY gene using HaeIII, MseI and RsaI restriction enzymes showed profiles indistinguishable from each other. This is the first study reporting E. camaldulensis as a new host species for “Ca. P. solani.”     

Land‐use changes influence the sporulation and survival of Phytophthora agathidicida,a lethal pathogen of New Zealand kauri (Agathis australis)

Phytophthora agathidicida is the accepted causal agent of dieback in remnant stands of long‐lived indigenous New Zealand kauri (Agathis australis) and poses a significant threat to the long‐term survival of this species. Little is known about the effect of key soil physicochemical characteristics on the growth of P. agathidicida. In this study, we investigated the growth of P. agathidicida in soils collected from adjacent areas under original kauri forest, short rotation pine (Pinus radiata) plantation forest and grazed pastures. A growth response assay was used to quantify asexual (sporangia) and sexual (oospore) spore counts over 8 days in soils sampled from each land‐use. Significantly higher numbers of sporangia (p < 0.001) and oospores (p < 0.01) were found in pasture and pine forest soil within 2 days of the growth assay trials, suggesting these soils may favour asexual/sexual reproduction in the early stages of P. agathidicida establishment compared to kauri forest soils. Additionally, oospore production significantly increased over 8 days in pine forest soil, suggesting that with an increase in inoculum loads, these soils potentially act as pathogen reservoirs. The soil physicochemical properties (e.g., pH, C and N, phosphorus content and electrical conductivity) investigated in this study did not significantly correspond to spore count data between land‐uses, suggesting that differences in growth response are driven by other edaphic factors not explored in the present study.     

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.     

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.