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.

In vitro antioxidant extracts evaluation from the residue of the Hevea brasiliensis seed

The antioxidants used in the food industry are essential to inhibit the formation of free radicals, preserving the existing properties in the different matrices. However, the insecurity of the synthetic antioxidants regarding human health propels search for natural substrates with potential antioxidant activity as an alternative to synthetic compounds. In this way, the work had as objective obtaining extracts from the seed pomace of the Hevea brasiliensis (rubber tree), relating the contents of flavonoids and total phenols in the application as an antioxidant. The methodology consisted of the extraction using four solvents, varying extractive methods, time, and seed concentrations. The antioxidant activity in vitro was evaluated by capturing the DPPH (2,2-diphenyl-1-picryl-hydrazil) radical. The optimized results demonstrate that the aqueous extracts produced in the Soxhlet in the concentrations of 85 g L−1 and retention time of 4 h reached 37.73 ± 1.69% in the antioxidant tests of the free radical DPPH capture, 1405.15 mg EAC 100 g−1 in the quantification of phenolic compounds and 223.34 mg 100 g−1 of total flavonoids. Thus, this work may contribute to the realization of studies and future research for characterization and identification concerning which phenolic compounds and flavonoids attribute the antioxidant characteristic to the extracts produced, enabling the discovery of products with high added value in the production chain. In addition, because the water used as a solvent showed greater antioxidant potential between the extracts, the non-toxic and environmentally friendly character is highlighted, allowing a wide variety of applications in the food industry.

Morphology and multi-gene phylogeny reveal a new fungal genus and species from Hevea brasiliensis latex in Yunnan, China

During a survey of rubber tree plantations in Yunnan Province, China, a fungus growing on natural rubber latex was collected and isolated. Maximum likelihood and Bayesian analyses of combined ITS, LSU, SSU, tef-α and rpb2 genes showed the isolated taxon belongs to Wiesneriomycetaceae and was well separated from closely related genera with high statistical support. It can be differentiated from other genera in Wiesneriomycetaceae by synnemata with catenate conidia that are cylindrical, 0–4 to multi-septate, often with a dark brown band at the septa. Morphology, phylogenetic analyses and pairwise homoplasy index (PHI) tests provide evidence that the new isolate is a distinct genus. The genus Heveicola is introduced, with H. xishuangbannaensis as the type species. A key to the genera in Wiesneriomycetaceae is also provided.

Differential Expression of lncRNAs and miRNAs Between Self-Rooting Juvenile and Donor Clones Unveils Novel Insight Into the Molecular Regulation of Rubber Biosynthesis in Hevea brasiliensis

The rubber tree (Hevea brasiliensis Muell. Arg.) is a tropical tree species that produce natural rubber. Self-rooted juvenile clones (SRJCs) are novel rubber tree planting materials developed through primary somatic embryogenesis. SRJCs have a higher rubber yield compared with donor clones (DCs). The molecular basis underlying increased rubber yield in SRJCs remains largely unknown. Here, the latex from SRJCs and DCs were collected for strand-specific and small RNA-seq methods. A total of 196 differentially expressed long noncoding RNAs (DELs), and 11 differentially expressed microRNAs were identified in latex between SRJCs and DCs. Targeted genes of DELs were markedly enriched for various biological pathways related to plant hormone signal transduction, photosynthesis, glutathione metabolism, and amino acids biosynthesis. DELs probably acted as cis-acting regulation was calculated, and these DELs relevant to potentially regulate rubber biosynthesis, reactive oxygen species metabolism, and epigenetic modification. Furthermore, the DELs acting as microRNA targets were studied. The interaction of microRNA and DELs might involve in the regulation of natural rubber biosynthesis.

Functional Identification of ICE Transcription Factors in Rubber Tree

ICE (inducer of CBF expression) is a positive regulator of cold signaling pathway in plants. Identification of ICE transcription factors is important for the sustainable development of the natural rubber planting industry in nontraditional regions where sudden cold waves often occur. In this study, five ICE genes were isolated from genome of rubber tree (Hevea brasiliensis Muell. Arg.) for analysing tolerance to cold stress. They shared an ICE-specific region in the highly conserved bHLH-ZIP domain and were localized in the nucleus. The HbICEs were different in transcript abundance and expression patterns in response to cold and drought stresses and among different rubber tree clones. Generally, the expression level of HbICEs was significantly higher in the cold-tolerant rubber tree clones than that in the cold-sensitive rubber tree clones. Overexpression of HbICE1, HbICE2, and HbICE4 significantly enhanced the cold tolerance of transgenic Arabidopsis and tobacco, which showed a significant increase in chlorophyll content and decrease in relative water content and conductivity at the early stage of cold stress in comparison with wild-type plants. Furthermore, overexpression of HbICE2 and HbICE4, but also HbICE1 enhanced drought tolerance in transgenic Arabidopsis. The cold tolerance of rubber tree clones is positively controlled by the expression level of HbICE1, HbICE2, and HbICE4.

First report of rubber tree wilt caused by Ceratocystis fimbriata in China

The rubber tree (Hevea brasiliensis) is an important economic resource for the rubber and latex industry. During November 2013 and June 2016, rubber trees showing typical wilt symptoms were found in Mengla, Xishuangbannan, Yunnan, China (N 21° 28', E 101° 33'). Symptomatic trees initially exhibited wilting of foliage on individual branches, then spread to the whole canopy, finally followed by death of the whole tree. Dark-blue to black discoloration was observed in the inner bark and affected xylem, a grayish layer of fungal growth and sporulation occasionally. The disease was detected on 20% of trees surveyed. The diseased tissues of three rubber trees were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA), and incubated at 25°C. After 7 days, a fungus was consistently observed growing from the tissue. Three single-spore isolates were obtained. In culture, colonies reaching 69 mm diam within 10 days, mycelium was initially white, then becoming celadon. After 5 days of perithecium formation, observed perithecia were black, globose (173.1 - 237.9 × 175.6 - 217.2 μm) and showed a long black neck (507.3 - 794.1 μm). Ascospore with outer cell wall forming a brim, hat-shaped at the tips of ostiolar hyphae (3.43 × 5.63 μm). Cylindrical endoconidia (10.5 - 39.7 × 3.5 - 6.6 μm) were hyaline. Chain of barrel-shaped conidia (7.2 - 9.5 × 4.1 - 6.2 μm) was found. Aleuroconidia were ovoid or obpyriform, and smooth (10.2 - 14.1 × 8.4 - 10.6 μm). Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata (Engelbrecht and Harrington 2005). The genomic DNA was extracted from isolates (XJm10-2-5, XJm8-2-5, XJm4) 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 KJ511488, KJ511485, KT963149) showed that the isolates were 100% homologous to those of the isolates on Punica granatum and Colocasia esculenta from China (GenBank accessions KT963152, MH793673) by BLAST analysis. Neighbor-joining phylogenetic analyse were performed using MEGA 6.06 based on ITS sequences (Fig. 1). Analyses showed that all isolates located on the same clade with all C. fimbriata with a high bootstrap support. Therefore, the fungus was identified as C. fimbriata based on morphology and molecular evidences. Pathogenicity of C. fimbriata isolated from this study was tested by inoculation of three one-year-old pot-grown (3L) seedlings of rubber tree. The soil of three seedlings was inoculated by drenching with 30 ml spore suspension (2.0 × 106 spores / ml). Three control plants were inoculated with 30 ml of sterile distilled water. The experiment was repeated three times. The plants were kept in a controlled greenhouse at 25°C and watered weekly. After the inoculation for one month, all the isolates produced typical wilt symptoms, while control plants showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. fimbriata according to the methods described above. The pathogenicity assay showed that C. fimbriata was pathogenic to rubber trees. C. fimbriata was first reported on rubber tree in Brazil (Albuquerque et al. 1972; Silveira et al. 1985). To the best of our knowledge, this is the first report of C. fimbriata causing wilt of rubber tree in China. This finding contributes to understanding the diversity of this pathogen, and it appears to be a significant threat to rubber trees in its ecosystem.

Reconstitution of Prenyltransferase Activity on Nanodiscs by Components of the Rubber Synthesis Machinery of the Para Rubber Tree and Guayule

Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity. The proteins HRT1, HRT2, and HRBP have been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Co-expression of HRT1 and HRBP in the presence of nanodiscs yielded marked polyisoprene synthesis activity. By contrast, neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP manifested such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (PaCPT1–3) manifested prenyltransferase activity only if co-expressed with PaCBP, the homolog of HRBP. Our results thus indicate that two heterologous subunits form the core prenyltransferase of the rubber biosynthetic machinery. A recently developed structure modeling program predicted the structure of such heterodimer complexes including HRT1/HRBP and PaCPT2/PaCBP. HRT and PaCPT proteins were also found to possess affinity for a lipid membrane in the absence of HRBP or PaCBP, and structure modeling implicated an amphipathic α-helical domain of HRT1 and PaCPT2 in membrane binding of these proteins.

Rubber tree and cassava: Biological features, practical use, ethnocultural traditions

Aim. To make a review of Hevea and Manihot, two related plant genera that have been used by humans for a long time, to analyze their biological features, to give information on their use, to present the most interesting materials associated with them. Main part. A review of Hevea and Manihot, useful plants in the tropics and subtropics, is carried out. Their characteristics are given, biological features are considered, and data on the places of origin, on the regions where they are cultivated are considered. Information on their use by people is presented: Hevea as a rubber-bearing, cassava as a food product. Special attention is paid to the cytogenetic features of Hevea and Manihot, information about the ancient polyploid origin of these plants. Conclusion. From ancient to the present time, mankind has been using in everyday life many valuable species of subtropical and tropical plants, including Hevea and Manihot, which are an integral part of ethnocultural traditions.