BackgroundFusarium circinatum is a pressing threat to the cultivation of many economically important pine tree species. Efforts to develop effective disease management strategies can be aided by investigating the molecular mechanisms involved in the host-pathogen interaction between F. circinatum and pine species. Pinus tecunumanii and Pinus patula are two closely related tropical pine species that differ widely in their resistance to F. circinatum challenge, being resistant and susceptible respectively, providing the potential for a useful pathosystem to investigate the molecular responses underlying resistance to F. circinatum. However, no genomic resources are available for P. tecunumanii. Pathogenesis-related proteins are classes of proteins that play important roles in plant-microbe interactions, e.g. chitinases; proteins that break down the major structural component of fungal cell walls. Generating a reference sequence for P. tecunumanii and characterizing pathogenesis related gene families in these two pine species is an important step towards unravelling the pine-F. circinatum interaction.ResultsEight reference based and 12 de novo assembled transcriptomes were produced, for juvenile shoot tissue from both species. EvidentialGene pipeline redundancy reduction, expression filtering, protein clustering and taxonomic filtering produced a 50 Mb shoot transcriptome consisting of 28,621 contigs for P. tecunumanii and a 72 Mb shoot transcriptome consisting of 52,735 contigs for P. patula. Predicted protein sequences encoded by the assembled transcriptomes were clustered with reference proteomes from 92 other species to identify pathogenesis related gene families in P. patula, P. tecunumanii and other pine species.ConclusionsThe P. tecunumanii transcriptome is the first gene catalogue for the species, representing an important resource for studying resistance to the pitch canker pathogen, F. circinatum. This study also constitutes, to our knowledge, the largest index of gymnosperm PR-genes to date.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5015-0) contains supplementary material, which is available to authorized users.

BackgroundPines are the most important tree species to the international forestry industry, covering 42 % of the global industrial forest plantation area. One of the most pressing threats to cultivation of some pine species is the pitch canker fungus, Fusarium circinatum, which can have devastating effects in both the field and nursery. Investigation of the Pinus-F. circinatum host-pathogen interaction is crucial for development of effective disease management strategies. As with many non-model organisms, investigation of host-pathogen interactions in pine species is hampered by limited genomic resources. This was partially alleviated through release of the 22 Gbp Pinus taeda v1.01 genome sequence (http://pinegenome.org/pinerefseq/) in 2014. Despite the fact that the fragmented state of the genome may hamper comprehensive transcriptome analysis, it is possible to leverage the inherent redundancy resulting from deep RNA sequencing with Illumina short reads to assemble transcripts in the absence of a completed reference sequence. These data can then be integrated with available genomic data to produce a comprehensive transcriptome resource. The aim of this study was to provide a foundation for gene expression analysis of disease response mechanisms in Pinus patula through transcriptome assembly.ResultsEighteen de novo and two reference based assemblies were produced for P. patula shoot tissue. For this purpose three transcriptome assemblers, Trinity, Velvet/OASES and SOAPdenovo-Trans, were used to maximise diversity and completeness of assembled transcripts. Redundancy in the assembly was reduced using the EvidentialGene pipeline. The resulting 52 Mb P. patula v1.0 shoot transcriptome consists of 52 112 unigenes, 60 % of which could be functionally annotated.ConclusionsThe assembled transcriptome will serve as a major genomic resource for future investigation of P. patula and represents the largest gene catalogue produced to date for this species. Furthermore, this assembly can help detect gene-based genetic markers for P. patula and the comparative assembly workflow could be applied to generate similar resources for other non-model species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2277-7) contains supplementary material, which is available to authorized users.

RNA-sequencing technology has been widely adopted to investigate host responses during infection with pathogens. Dual RNA-sequencing (RNA-seq) allows the simultaneous capture of pathogen-specific transcripts during infection, providing a more complete view of the interaction. In this review, we focus on the design of dual RNAseq experiments and the application of downstream data analysis to gain biological insight into both sides of the interaction. Recent literature in this area demonstrates the power of the dual RNA-seq approach and shows that it is not limited to model systems where genomic resources are available. Sequencing costs continue to decrease and single cell transcriptomics is becoming more feasible. In combination with proteomics and metabolomics studies, these technological advances are likely to contribute to our understanding of the temporal and spatial aspects of dynamic plant-pathogen interactions. A dual approach in plantaThe interaction between plants and pathogens is an active and dynamic process that can be likened to a duel. Plants have complex defence mechanisms that can be rendered ineffective when pathogens interfere with one of the various processes required for host defence. These processes include penetration resistance, recognition by Pattern Recognition Receptors (PRRs), phytohormone signalling pathways, secretory pathways, secondary metabolite production, and plant cell death (Dou and Zhou, 2012). Until recently, transcriptomic approaches have been applied in the host and pathogen separately to obtain the gene expression profile of each organism and gain insight into infection biology or host defence mechanisms.RNA sequencing (RNA-seq) is a powerful technology that does not rely on any prior knowledge of transcripts and can generate vast quantities of data with much smaller costs involved than for older techniques such as microarrays (Pareek et al., 2011;Wilhelm and Landry, 2009). An advantage of RNA-seq in the field of plant-pathogen interactions is that both plant and pathogen transcripts can be detected simultaneously and accurately in the same sample. This tactic, known as dual RNAseq, in planta RNA-seq, simultaneous RNA-seq, or comparative RNA-seq, is a relatively new technique both in the plant and medical fields. In plants, it allows for the study of plant-pathogen interactions in herbaceous crops Kunjeti et al., 2012;Lowe et al., 2014) as well as trees (Hayden et al., 2014;Liang et al., 2014;Teixeira et al., 2014). This review outlines technical considerations for dual RNA-seq experiments, summarizes recent insights drawn from such approaches in plant-pathogen interactions, and provides an

Fusarium circinatum poses a serious threat to many pine species in both commercial and natural pine forests. Knowledge regarding the molecular basis of pine-F. circinatum host-pathogen interactions could assist efforts to produce more resistant planting stock. This study aimed to identify molecular responses underlying resistance against F. circinatum. A dual RNA-seq approach was used to investigate host and pathogen expression in F. circinatum challenged Pinus tecunumanii (resistant) and Pinus patula (susceptible), at three- and seven-days post inoculation. RNA-seq reads were mapped to combined host-pathogen references for both pine species to identify differentially expressed genes (DEGs). F. circinatum genes expressed during infection showed decreased ergosterol biosynthesis in P. tecunumanii relative to P. patula. For P. tecunumanii, enriched gene ontologies and DEGs indicated roles for auxin-, ethylene-, jasmonate- and salicylate-mediated phytohormone signalling. Correspondingly, key phytohormone signaling components were down-regulated in P. patula. Key F. circinatum ergosterol biosynthesis genes were expressed at lower levels during infection of the resistant relative to the susceptible host. This study further suggests that coordination of phytohormone signaling is required for F. circinatum resistance in P. tecunumanii, while a comparatively delayed response and impaired phytohormone signaling contributes to susceptibility in P. patula.

Background: Fusarium circinatum, the causal agent of pitch canker disease, poses a serious threat to several Pinus species affecting plantations and nurseries. Although Pinus pinaster has shown moderate resistance to F. circinatum, the molecular mechanisms of defense in this host are still unknown. Phytohormones produced by the plant and by the pathogen are known to play a crucial role in determining the outcome of plant-pathogen interactions. Therefore, the aim of this study was to determine the role of phytohormones in F. circinatum virulence, that compromise host resistance. Results: A high quality P. pinaster de novo transcriptome assembly was generated, represented by 24,375 sequences from which 17,593 were full length genes, and utilized to determine the expression profiles of both organisms during the infection process at 3, 5 and 10 days post-inoculation using a dual RNA-sequencing approach. The moderate resistance shown by Pinus pinaster at the early time points may be explained by the expression profiles pertaining to early recognition of the pathogen, the induction of pathogenesis-related proteins and the activation of complex phytohormone signaling pathways that involves crosstalk between salicylic acid, jasmonic acid, ethylene and possibly auxins. Moreover, the expression of F. circinatum genes related to hormone biosynthesis suggests manipulation of the host phytohormone balance to its own benefit. Conclusions: We hypothesize three key steps of host manipulation: perturbing ethylene homeostasis by fungal expression of genes related to ethylene biosynthesis, blocking jasmonic acid signaling by coronatine insensitive 1 (COI1) suppression, and preventing salicylic acid biosynthesis from the chorismate pathway by the synthesis of isochorismatase family hydrolase (ICSH) genes. These results warrant further testing in F. circinatum mutants to confirm the mechanism behind perturbing host phytohormone homeostasis.

Long-lived tree species are subject to attack by various pests and pathogens during their lifetime. This problem is exacerbated by climate change, which may increase the host range for pathogens and extend the period of infestation by pests. Plant defences may involve preformed barriers or induced resistance mechanisms based on recognition of the invader, complex signalling cascades, hormone signalling, activation of transcription factors and production of pathogenesis-related (PR) proteins with direct antimicrobial or anti-insect activity. Trees have evolved some unique defence mechanisms compared with well-studied model plants, which are mostly herbaceous annuals. The genome sequence of Eucalyptus grandis W. Hill ex Maiden has recently become available and provides a resource to extend our understanding of defence in large woody perennials. This review synthesizes existing knowledge of defence mechanisms in model plants and tree species and features mechanisms that may be important for defence in Eucalyptus, such as anatomical variants and the role of chemicals and proteins. Based on the E. grandis genome sequence, we have identified putative PR proteins based on sequence identity to the previously described plant PR proteins. Putative orthologues for PR-1, PR-2, PR-4, PR-5, PR-6, PR-7, PR-8, PR-9, PR-10, PR-12, PR-14, PR-15 and PR-17 have been identified and compared with their orthologues in Populus trichocarpa Torr. & A. Gray ex Hook and Arabidopsis thaliana (L.) Heynh. The survey of PR genes in Eucalyptus provides a first step in identifying defence gene targets that may be employed for protection of the species in future. Genomic resources available for Eucalyptus are discussed and approaches for improving resistance in these hardwood trees, earmarked as a bioenergy source in future, are considered.

We performed gene and genome targeted SNP discovery towards the development of a genome-wide, multispecies genotyping array for tropical pines. Pooled RNA-seq data from shoots of seedlings from five tropical pine species was used to identify transcript-based SNPs resulting in 1.3 million candidate Affymetrix SNP probe sets.

The purpose of the present study is to evaluate the influence of drying air temperature on the essential oil content of sliced Melaleuca alternifólia Cheel. Three samples were taken randomly from each treatment and analyzed when still fresh in order to determine the moisture and essential oil contents. The samples were then cooled in the B.O.D at 2 °C till completion of the drying process. For the application of drying tests, an updraft fixed-bed dryer was used equipped with a liquefied petroleum gas (LPG) burner to heat up the drying air. The air velocity was 0.5  0.03 ms -1 and the relative humidity at the dryer entrance was 65%. The drying air temperatures were 40, 50, 60, 70 and 80°C. Three replications of the drying tests were done; where 500 g of the sliced plant cut into 5 cm thick layers were used. The essential oil was extracted in three hours by means of a hydro-distillation method. No statistical differences were detected between the drying treatments; however, when compared to the fresh plant, a reduction of 18.19% in oil content was observed as compared to the dried samples under different temperatures. INFLUÊNCIA DA TEMPERATURA DO AR DE SECAGEM NO TEOR DE ÓLEO ESSENCIAL DE Melaleuca alternifolia Cheel RESUMONo presente trabalho foi avaliada a influência da temperatura do ar de secagem sobre a quantidade do óleo essencial de Melaleuca alternifólia Cheel picada e tomadas, aleatoriamente, três amostras de cada tratamento as quais foram analisadas ainda frescas, avaliando-se o teor de água e o teor de óleo. Depois de retiradas as amostras o material foi resfriado em câmara climatizada a 2°C. Para a realização dos testes de secagem utilizou-se um secador de leito fixo, com fluxo de ar ascendente, equipado com um queimador de gás liquefeito de petróleo (GLP) para aquecimento do ar de secagem; a velocidade média do ar era de 0,5  0,03 m s -1 e sua umidade relativa média de entrada era de 65%. As temperaturas do ar de secagem foram 40, 50, 60, 70 e 80°C. Para cada teste de secagem foram utilizados aproximadamente 500 g de folhas trituradas, perfazendo uma camada de 5 cm de espessura, além de realizados três testes por tratamento. O óleo foi extraído por hidrodestilação, por três horas. Não houve diferença estatística entre os tratamentos de secagem; entretanto, quando comparados com a planta fresca verificaram-se redução da quantidade de óleo de 18,19% em relação ao material secado nas diferentes temperaturas.