First report of Eucalyptus wilt caused by Chalaropsis thielavioides in China

Eucalyptus species are widely planted in the tropics and subtropics, and eucalyptus is among the most important cash crop in Southern China. One of the most important diseases on eucalyptus is Ceratocystis wilt, caused by the fungus Ceratocystis fimbriata Ellis & Halst., and the genus name Chalaropsis has been proposed for anamorphs of Ceratocystis species (de Beer et al. 2014). During April 2018, severely infected Eucalyptus robusta trees were observed in Kunming, Yunnan Province, China. Symptomatic trees initially exhibited yellowing and wilting of foliage on individual branches, then spread to the whole canopy, sometimes followed by death of the whole tree. Reddish-brown to dark-brown discoloration in the woody xylem of affected trees, sometimes a grayish white layer of fungal growth may be seen. The disease was observed on 16% of trees surveyed. The base of trunks with typical symptoms were collected, then the discolored xylem tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, a fungus was consistently observed growing from the tissue. Three isolates were obtained. In culture, colonies reaching 54mm diam within 15 days, mycelium initially white, then becoming celadon. Endoconidia unicellular, smooth, cylindrical, straight, biguttulate, 11.21 - 32.26 × 4.12 - 5.25 μm. Phialides produced on short, septate, aerial hyphae, lageniform and chain of phialoconidia (3.62 - 5.89 × 31.39 - 65.76 μm) were also observed. Chlamydospores (11.45 - 14.26 × 10.06 - 12.22 μm) were single, dark, thick-walled. Morphological characteristics of the fungus were consistent with the description of Chalaropsis thielavioides (Paulin-Mahady et al. 2002). The two of three isolates were used for molecular identification and genomic DNA was extracted from isolates (EKY2-2-1, EKY2-2-2) using the chelex-100 method (Xu et al. 2020). The ITS region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of ITS sequence data (GenBank accessions MW242701, MW242702) showed that the isolates were 99% - 100% homologous to isolates of C. thielavioides from Hevea rubber, Monstera deliciosa L. and ants in China and Rosa sp. in Australia (GenBank accessions KT963172, KJ511482, KT963173 and KX954598) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis were performed using MEGA 6.06 based on ITS sequences (Fig 1), the evolutionary distances were computed using the Maximum Composite Likelihood method. Analyses showed that both isolates (EKY2-2-1, EKY2-2-2) located on the same clade with all C. thielavioides, and clustered with the C. thielavioides strains with high bootstrap support (97% - 100%). Therefore, the fungus was identified as C. thielavioides based on morphology and molecular evidences. Pathogenicity of C. thielavioides was tested by inoculation of six one-year-old pot grown Eucalyptus citriodora seedlings. The sterilized soil of six seedlings was inoculated by drenching with 20 ml spore suspension (2.0 × 106 spores / ml). Control plants were inoculated with 20 ml of sterile distilled water. The seedlings were kept in a controlled greenhouse at 25°C and watered weekly. After one month incubation, all the isolates produced wilt symptoms, whereas control trees showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. thielavioides according to the methods described above, and no fungal growth was observed in the controls, thus satisfying Koch's postulates. Although wilt and canker caused by Ceratocystis fimbriata on eucalyptus have been previously reported in Brazil, Uruguay, Uganda, China and Pakistan (Ferreira et al. 1999; Li et al. 2014; Alam et al. 2017), eucalyptus wilt caused by C. thielavioides has not been reported anywhere. Also, wilt of rubber tree and postharvest rot on carrot caused by C. thielavioides have been reported (Li et al. 2021; Xu et al. 2020). To our knowledge, this is the first report of eucalyptus wilt caused by C. thielavioides in China.

HYPSOMETRY AND VOLUMETRY OF Eucalyptus urograndis IN A CROP-FOREST INTEGRATION SYSTEM

Due to the positive impact that the eucalyptus species has on the Brazilian economy, it is currently the most used forest essence. The objective of this work was to evaluate different hypsometric and volumetric models for Eucalyptus urograndis clones (Eucalyptus urophylla S.T. Blak and Eucalyptus grandis W. Hill ex Maiden) in a Crop-Forest Integration (CFI) system. The trees were evaluated at 7 years of age and arranged in double rows, occupying 20.76% of the total system area. The individuals were subjected to rigorous volumetric cubing according to the Smalian method at intervals of one meter up to full height. The following models were evaluated for the collected height data: Linear, Trorey, Stofels, Curtis, Henriksen, Prodan, Chapman & Richards, Petterson and Bailey & Clutter. Furthermore, the Spurr, Hohenald-Krenn, Stoate, Schumacher Hall, Meyer, Husch, Ogaya and Takata models were used for volume data. The results were determined through the coefficient of determination (R2), standard error of the estimate in percentage (Syx%), significance of the regression coefficients (𝛽) and graphical distribution. The hypsometric model which best fit the database among tested models was the Prodan equation, with a coefficient of determination (R²) of 0.89, while the best result for volumetric models was found using the Meyer model, with a coefficient of determination (R²) of 0.99. All evaluated models were efficient in estimating the height and volume of the Crop-Forest Integration (CFI) system, thus demonstrating that GG100 eucalyptus is a good option in integrated systems.

Gene expression programs during callus development in tissue culture of two Eucalyptus species

Background Eucalyptus is a highly diverse genus of the Myrtaceae family and widely planted in the world for timber and pulp production. Tissue culture induced callus has become a common tool for Eucalyptus breeding, however, our knowledge about the genes related to the callus maturation and shoot regeneration is still poor. Results We set up an experiment to monitor the callus induction and callus development of two Eucalyptus species - E. camaldulensis (high embryogenic potential) and E. grandis x urophylla (low embryogenic potential). Then, we performed transcriptome sequencing for primary callus, mature callus, shoot regeneration stage callus and senescence callus. We identified 707 upregulated and 694 downregulated genes during the maturation process of the two Eucalyptus species and most of them were involved in the signaling pathways like plant hormone and MAPK. Next, we identified 135 and 142 genes that might play important roles during the callus development of E. camaldulensis and E. grandis x urophylla, respectively. Further, we found 15 DEGs shared by these two Eucalyptus species during the callus development, including Eucgr.D00640 (stem-specific protein TSJT1), Eucgr.B00171 (BTB/POZ and TAZ domain-containing protein 1), Eucgr.C00948 (zinc finger CCCH domain-containing protein 20), Eucgr.K01667 (stomatal closure-related actinbinding protein 3), Eucgr.C00663 (glutaredoxin-C10) and Eucgr.C00419 (UPF0481 protein At3g47200). Interestingly, the expression patterns of these genes displayed “N” shape in the samples. Further, we found 51 genes that were dysregulated during the callus development of E. camaldulensis but without changes in E. grandis x urophylla, such as Eucgr.B02127 (GRF1-interacting factor 1), Eucgr.C00947 (transcription factor MYB36), Eucgr.B02752 (laccase-7), Eucgr.B03985 (transcription factor MYB108), Eucgr.D00536 (GDSL esterase/lipase At5g45920) and Eucgr.B02347 (scarecrow-like protein 34). These 51 genes might be associated with the high propagation ability of Eucalyptus and 22 might be induced after the dedifferentiation. Last, we performed WGCNA to identify the co-expressed genes during the callus development of Eucalyptus and qRT-PCR experiment to validate the gene expression patterns. Conclusions This is the first time to globally study the gene profiles during the callus development of Eucalyptus. The results will improve our understanding of gene regulation and molecular mechanisms in the callus maturation and shoot regeneration.

Sources of phenotypic variation of wood density and relationships with mean growth in two Eucalyptus species in Argentina

Aims of the study: To describe the radial patterns of wood density, and to identify their main sources of variation, and the potential tradeoffs with mean tree growth, in two Eucalyptus species. Area of study: Mesopotamian (Corrientes and Entre Ríos provinces) and Pampean region (Buenos Aires province) of Argentina. Materials and methods: Eucalyptus grandis and Eucalyptus viminalis, growing in genetic trials installed in two sites per species were studied. X-ray wood microdensity profiles were developed from core samples. Each profile was proportionally divided in 10 sections. Mean, maximum, minimum and the standard deviation of wood density, for each section were computed. Mean annual growth was used to study the relationships with wood microdensity variables. A linear mixed-effects model computed the significance of different sources of phenotypic variation. Pearson´s correlation computed the relationships between variables. Main results: The pattern of radial variation in E. grandis showed a decrease in wood density from pith to bark, mainly due to the decrease in minimum wood density, while in E. viminalis, wood density increased towards the outer wood. In both species, the standard deviation of the wood density increased along the radial profile from pith to bark. Significant variation in wood density was explained by site, provenance and clone/family effects. In E. grandis mean, maximum and minimum wood density were negatively correlated with mean growth, whereas in E. viminalis correlations were positive but close to zero. Research highlights: Both the pattern of radial variation of wood density and the relationship between wood density and mean growth were different in the studied Eucalyptus species, and they varied within species depending on the site they were growing and genetic provenance.

Expression Patterns and Regulation of Non-Coding RNAs during Synthesis of Cellulose in Eucalyptus grandis Hill

Cellulose, an essential structural component in the plant cell wall and a renewable biomass resource, plays a significant role in nature. Eucalyptus’s excellent timber tree species (including Eucalyptus grandis Hill) provide many raw materials for the paper and wood industries. The synthesis of cellulose is a very complex process involving multiple genes and regulated by various biological networks. However, research on regulating associated genes and non-coding RNAs during cellulose synthesis in E. grandis remains lacking. In this study, the wood anatomical characteristics and chemical indexes of E. grandis were analyzed by taking three different parts (diameter at breast height (DBH), middle and upper part of the trunk) from the main stem of E. grandis as raw materials. The role of non-coding RNAs (Long non-coding RNA, lncRNA; Micro RNA, miRNA; Circle RNA, circRNA) on regulating candidate genes was presented, and the network map of ceRNA (Competing endogenous RNA) regulation during wood cellulose biosynthesis of E. grandis was constructed. The transcriptome sequencing of nine samples obtained from the trunk of the immature xylem in E. grandis at DBH, middle and upper parts had a 95.81 G clean reading, 57,480 transcripts, 7365 lncRNAs, and 5180 circRNAs. Each sample had 172–306 known miRNAs and 1644–3508 new miRNAs. A total of 190 DE-lncRNAs (Differentially expressed long non-coding RNAs), 174 DE-miRNAs (Differentially expressed micro RNAs), and 270 DE-circRNAs (Differentially expressed circle RNAs) were obtained by comparing transcript expression levels. Four lncRNAs and nine miRNAs were screened out, and the ceRNA regulatory network was constructed. LncRNA1 and lncRNA4 regulated the genes responsible for cellulose synthesis in E. grandis, which were overexpressed in 84K (Populus Alba × Populus glandulosa) poplar. The cellulose and lignin content in lncRNA4-oe were significantly higher than wild type 84K poplar and lncRNA1-oe. The average plant height, middle and basal part of the stem diameter in lncRNA4-oe were significantly higher than the wild type. However, there was no significant difference between the growth of lncRNA1-oe and the wild type. Further studies are warranted to explore the molecular regulatory mechanism of cellulose biosynthesis in Eucalyptus species.

Closely-related tree species with overlapping ranges exhibit divergent adaptation to climate

With global climate change shifting and altering temperature and precipitation regimes, the ability of natural forest stands to persist in their local environments are being challenged. For many taxa, particularly among long lived tree species, the potential to respond is underpinned by genetic and trait diversity and may be limited. We sampled 326 and 366 individuals of two widely distributed and closely-related red gum Eucalyptus species (E. blakelyi and E. tereticornis) from across their entire Australian range. We identified putatively adaptive variants associated within genes of key biological processes for both species. We mapped the change of allele frequencies of two hierarchical gene ontology groups shared by both species across geography and climate and predict genomically vulnerable regions under a projected 2070 climate scenario. Regions of potential vulnerability to decline under future climate differed between species and may be applied to guide conservation and restoration strategies. Our study indicated that some populations may contain the adaptive genomic variation necessary for these species to persist through climate change, while others may benefit from the adaptive variation of those populations to enhance resilience.

Anthocyanins, Phenolic Compounds, and Antioxidants from Extractions of Six Eucalyptus Species

The leaves of Eucalyptus have multiple biological activities such as antimicrobial, antiseptic, antioxidant, and antifungal. A Soxhlet extraction, SLE, and HD were used to obtain extracts from the leaves of six Eucalyptus species, E. globulus, E. oblicua, E. pavaflora, E. camaldulensis, E. viminalis, and E. nitens, and to study their antioxidant capacity. Solvents such as acetone, dichloromethane, ethanol, hexane, methanol, and water were used to study how polarity influences extraction yields. The SLE method achieved higher or similar yields, depending on the species and its composition, than the Soxlet method at a temperature of 50 °C. The highest yields were obtained with E. viminalis with methanol (42.5 wt.%), the highest phenolic content was obtained with E. nitens with methanol (124.17 mg GAE/g of extract), and the highest anthocyanin levels obtained were with E. nitens with hexane (5.05 mg CC/g of extract). E. nitens obtained almost five times more phenolic content than E. globulus; therefore, it is the most promising species. The high content of the compounds analysed confirm the good potential of these species to obtain value-added compounds. Our results demonstrate that the differences in the extract contents depend on the polarity of the solvents used. In addition, the use of these species will reduce the residue in the forest, which is greatly beneficial.

Leaf 13C and 15N composition shedding light on easing drought stress through partial K substitution by Na in eucalyptus species

This work aimed to investigate the partial K-replacement by Na supply to alleviate drought-induced stress in Eucalyptus species. Plant growth, leaf gas exchange parameters, water relations, oxidative stress (H2O2 and MDA content), chlorophyll concentration, carbon (C) and nitrogen (N) isotopic leaf composition (δ13C and δ15N) were analyzed. Drought tolerant E. urophylla and E. camaldulensis showed positive responses to the partial K substitution by Na, with similar dry mass yields, stomatal density and total stomatal pore area relative to the well K-supplied plants under both water conditions, suggesting that 50% of the K requirements is pressing for physiological functions that is poorly substituted by Na. Furthermore, E. urophylla and E. camaldulensis up-regulated leaf gas exchanges, leading to enhanced long-term water use efficiency (WUEL). Moreover, the partial K substitution by Na had no effects on plants H2O2, MDA, δ13C and δ15N, confirming that Na, to a certain extent, can effectively replace K in plants metabolism. Otherwise, the drought-sensitive E. saligna species was negatively affected by partial K replacement by Na, decreasing plants dry mass, even with up-regulated leaf gas exchange parameters. The exclusive Na-supplied plants showed K-deficient symptoms and lower growth, WUEL, and δ13C, besides higher Na accumulation, δ15N, H2O2 and MDA content.

Long-Term Effects of Forest Plantation Species on Chemical Soil Properties in Southern Rwanda

Understanding the long-term effects of tree species on soil properties is crucial for the development of forest restoration policies in relation to the choice of species that meet both environmental and local livelihood needs. This study was performed in the Arboretum of Ruhande, Southern Rwanda, where monocultures of 148 deciduous and 56 conifer species have been established in 0.25 ha replicated plots from 1933 onwards. We investigated the effects of six exotic and two native tree species planted in monoculture plots and native species mixed within one self-regenerated plot on soil properties in two layers (0–5 cm and 5–10 cm depth). We measured general soil properties (pH, SOM, exchangeable base cations) and water-soluble C and N as a proxy for soil functioning. Changes in soil properties were observed in the upper soil layer for all tree species. Planting Eucalyptus species caused soil acidification, whereas soil exchangeable cations and pH were higher under native species (Entandrophragma excelsum and Polyschias fulva) and mixed native species. The effects of tree species were more pronounced for hot water-extractable C and N than for other soil properties. Their analyses could be used for detecting changes in soil functioning linked to vegetation types.