Bark Tissues Research Articles

Molecular genetic analysis of wood and bark tissue vaccinations of different-type birch

Molecular genetic analysis of wood and bark tissue vaccinations of different-type birch

Spatiotemporal diversity of bacterial endophyte microbiome of mandarin (Citrus reticulata) in the northern Persian Gulf and its HCN production and N2 fixation.

Endophytes are symbionts that live in healthy plants and potentially improve the health of plant holobionts. Here, we investigated the bacterial endophyte community of Citrus reticulata grown in the northern Persian Gulf. Bacteria were isolated seasonally from healthy trees (root, stem, bark, trunk, leaf, and crown tissues) in four regions of Hormozgan province (i.e., Ahmadi, Siyahoo, Sikhoran, Roudan), a subtropical hot region in Iran. A total of 742 strains from 17 taxa, 3 phyla, and 5 orders were found, most of which belonged to Actinobacteria (Actinobacteriales) as the dominant group, followed by Firmicutes (Bacillales), Proteobacteria (Sphingomonadales, Rhizobiales), and Cyanobacteria (Synechoccales). The genera included Altererythrobacter, Arthrobacter, Bacillus, Cellulosimicrobium, Curtobacterium, Kocuria, Kytococcus, Methylopila, Mycobacterium, Nocardioides, Okiabacterium, Paracraurococcus, and Psychrobacillus. The most frequently occurring species included Psychrobacillus psychrodurans, Kytococcus schroetri, and Bacillus cereus. In addition, the overall colonization frequency and variability of endophytes were higher on the trunks. The leaves showed the lowest species variability in all sampling periods. The frequency of endophyte colonization was also higher in summer. The Shannon-Wiener (H') and Simpson indices varied with all factors, i.e., region, season, and tissue type, with the maximum in Roudan. Furthermore, 52.9% of the strains were capable of nitrogen fixation, and 70% produced antagonistic hydrogen cyanide (HCN). Thus, C. reticulata harbors a variety of bioactive bacterial endophytes that could be beneficial for host fitness in such harsh environments.

Relationship between the distribution of the pinewood nematode (Bursaphelenchus xylophilus) and the development of xylem embolism in the stems of Japanese black pine (Pinus thunbergii) seedlings monitored by magnetic resonance imaging.

The development of xylem embolism in 1-year-old stems of Japanese black pine (Pinus thunbergii) seedlings was monitored by compact magnetic resonance imaging (MRI) after inoculation with the pinewood nematode (Bursaphelenchus xylophilus). In parallel, the nematode distribution and population structure in the stems were examined by isolating the nematodes using the Baermann funnel technique. The vertical length and volume of massive embolisms in each seedling were strongly correlated with the maximum relative embolized area (REA) in stem cross-sections. Embolism development and nematode reproduction were not restricted to the inoculation site, as any portion of the stem could be the initial point of a population burst. The nematode population in the stem xylem was strongly correlated with the REA and with the circumferential proportion of cambial death in cross-sections monitored by MRI. The proportion of second-stage juveniles was also correlated with the REA in the xylem. In contrast, the nematode population in bark tissue was not correlated with either the REA or cambial death. These results suggested that nematode reproduction in the cambial zone is the key step in pine wilt disease, and second-stage juveniles were suggested to induce massive embolisms in the advanced stage of the disease.

Harnessing Endophytic Fungi from Terminalia chebula: A Dual Approach to Antimicrobial and Phytochemical Exploration

Endophytic fungi isolated from Terminalia chebula exhibit potent antimicrobial activity, showcasing their potential to develop novel therapeutic agents. Additionally, these fungi produce diverse phytochemicals, which may contribute to their medicinal properties and offer new avenues for natural product research. This research study investigated the endophytic fungi isolated from T. chebula and assessed their antimicrobial and phytochemical potential. A total of 28 endophytic fungal species were isolated from leaf and bark tissues. Antimicrobial assays revealed significant inhibitory effects against pathogenic fungi Candida albicans - MTCC 183 & Saccharomyces cerevisiae - MTCC 174 and bacteria Escherichia coli - MTCC 2939, & Bacillus subtilis - MTCC 1305, with endophytic Alternaria sp. exhibiting maximum inhibition zone for antifungal activity and endophytic Aspergillus flavus demonstrating potent activity for their antibacterial analysis. A wide array of phytochemical compounds like alkaloids, flavonoids, phenols, steroids, terpenoids, tannin, saponin, etc, were observed whereas, phytochemical screening indicated the presence of alkaloids and flavonoids in all selected endophytes. These findings suggest that T. chebula harbors a rich endophytic community with the potential for producing novel antimicrobial compounds.

Feasibility and potential of terahertz spectral and imaging technology for Apple Valsa canker detection: A preliminary investigation

Apple Valsa canker (AVC) caused by the Ascomycete Valsa mali, seriously constrains the production and quality of apple fruits. The symptomless incubation characteristics of Valsa mali make it highly challenging to detect AVC at an early infection stage. After infecting the wound of apple bark, the pathogenic hyphae of AVC will expand and colonize the phloem tissue. Meanwhile, various enzymes and toxic substances released by hyphae cause the decomposition of cellulose and lignin, and the generation of poisonous secondary metabolites in bark tissue. However, these early symptoms of AVC are invisible from the bark’s appearance. Fortunately, Terahertz Spectral Imaging (ThzSI) technology with the advantage of penetrating, and fingerprinting is promising for detecting hidden or slight symptoms of the fungal infection. This study is a preliminary investigation of terahertz frequency-domain spectra for AVC in the early stage of infection. Healthy and two-week-infected apple tree branches were prepared for capturing ThzS images, and the spectral data were preprocessed by Multivariate scattering correction (MSC), Savitzky-Golay convolution smoothing (SG), and standard normal variate (SNV) respectively to remove data noise and improve data quality. Principal component analysis (PCA), competitive adaptive reweighted sampling (CARS), and random frog (RFROG) were employed to extract the spectral feature bands to eliminate redundant data and improve computational efficiency. Machine learning models were established based on the spectral features to detect AVC at an early infection stage, where 11 of them exhibited the best performance with F1-score of 99.72%. To further explore disease information in spatial spectra, imaging data were acquired using terahertz imaging technology. Based on imaging data, pseudo-color imaging, histogram equalization, and Otsu segmentation were employed to visualize early infection areas in apple barks. Furthermore, histogram feature (HF), shape feature (SF), and local binary pattern (LBP) extracted from terahertz spectral images were utilized to establish the SVM, RF, and KNN models. HF-SF-KNN and HF-SF-LBP-KNN with the best performance achieved F1-score of 98.82%. This study presents a preliminary application of terahertz spectral and imaging technology for early-stage AVC detection and demonstrates its feasibility. Additionally, it provides a new way to detect AVC, which expands the application of ThzSI technology in tree disease detection in orchards and lays the foundation for further research.

Herbicide Stress Inducesbeetle Oviposition on Red Maples.

Flatheaded borers (FHB; Chrysobothris spp.), are woodboring-beetles that lay their eggs in the bark and cambium of deciduous trees in North America. Females often target stressed host-plants for oviposition. The reason why is unknown; however, stressed plants often suffer various induced phytochemical changes that may enhance larval infestation success depending on the stressor such as induced upregulation of defenses, reallocation of nutrients, and changes to volatile organic compound (VOC) emissions. To understand attraction of FHB to specific stress-induced changes, we analyzed phytochemical changes associated with stress treatments and attractiveness maple trees to FHB. Trees were stressed by: (1) chemical stress (pelargonic acid herbicide), (2) physical stress (physically removing leaves), and (3) physical stress (removing portions of bark near the root crown). After reflush of defoliated trees, bark tissues where FHB larvae feed were analyzed for nutritional changes (carbon and nitrogen), anti-nutritive changes (polyphenols and tannins) and emissions of foliar VOCs. At the end of the growing season, trees were assessed for FHB larval presence and oviposition attempts. There were more larvae and oviposition attempts on trees stressed by herbicide application. Compared to other treatments, herbicide-stressed trees had greater nitrogen and total polyphenol concentrations. Greater nitrogen may play a role in the fitness of feeding larvae, and the greater polyphenol concentration may stimulate female oviposition in the herbicide stressed trees. Females may be able to locate the herbicide-stressed trees by using volatile cues such as increases in limonene, α-farnesene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and hexenyl acetate.

First report of Phytopythium vexans causing gummosis and root rot of Khasi Mandarin (Citrus reticulata Blanco) in north eastern states of India.

Khasi mandarin (Citrus reticulata Blanco) is the most economically important crop among the citrus growing region in the north-eastern India (Singh et al. 2016). An extensive survey was conducted to identify the causal agent of citrus root rot and gummosis in north eastern states (Meghalaya, Tripura, Manipur, Arunachal Pradesh, Sikkim, Nagaland and Assam) of India during October 2021-23. The gummosis disease incidence ranged from 5 to 95 % in 10 to 25 years old Khasi mandarin plants showing relatively more chronic symptoms on mature trees. Yellowing and dropping of leaves, twigs die back, gum oozing from infected bark and loss of feeder roots were the typical symptoms of the disease. Infected bark tissue and young lemon leaf baits in rhizosphere soil were plated on corn meal agar medium supplemented with pimaricin (10 µg/ml), ampicillin (250 µg/ml), rifamycin (10 µg/ml) and 300µg/ml carbendazim and incubated at 26℃. Fifty isolates were purified and maintained on Carrot agar medium. These isolates showed similar cultural and morphological characteristics. Two representative isolates from Arunachal Pradesh (AP21 and AP26) were selected for further experiments and deposited to Indian Type culture collection (ITCC), New Delhi with accession no. 9156 and 9157 respectively. The colonies were fast growing, showing rosette pattern along with whitish blooming mycelium appearance with no visual sporulation at the surface. The hyphae were coenocytic with initially right-angled branching. Sporangia were globose or sub globose and papillated. Oogonia were smooth and globose (16.29-21.09 µm) in diameter. Antheridia were irregular, cylindrical and broadly attached to oogonia. Empty sporangia were also observed. Multilocus phylogenetic analysis using internal transcribed spacer region (Das et al. 2011), β tubulin (Blair et al. 2008) and Cytochrome oxidase II gene (Noireung et al. 2020) showed that these isolates formed a stable clade with Phytopythium vexans (CBS119.80) sequence retrieved from NCBI database. BLAST analysis showed that ITS sequence of AP21 (OQ372986) and AP26 (OQ381083) had >99 % identity with P. vexans isolate NS-3 (ON533631). Further, BLAST analysis of β tubulin (AP21 OQ446053, AP26 OR405377) and Cox II gene (AP21 OQ473414, AP26 OR552422) sequences showed that our Indian isolates showed >99 % similarity with P. vexans voucher strain CBS119.80. To fulfil Koch's postulates, Khasi mandarin (Citrus reticulata) seedlings were inoculated by adding 100 ml zoospore suspension of P. vexans (1x105 spores/ml) in sterilized soil (Thao et al. 2020). The experiment was carried out in triplicate. Yellowing of leaves and leaf drop were observed 7 days post inoculation while 30 days post inoculation, treated plants started showing symptoms of root rot, including mild root decay. No symptoms were observed in control treatment. The pathogen was reisolated from symptomatic roots and confirmed through colony and sporangium morphology. Recently, it was reported that P. vexans is associated with apple and pear decline in the Saiss plain of Morocco (Jabiri et al. 2021), root rot on mandarin in Thailand (Noireung et al. 2020) and on Durian in Vietnam (Thao et al. 2020). As per our knowledge, this is the first report of P. vexans causing root rot and gummosis in Khasi mandarin from north eastern states of India. This finding is significantly important for the development of a successful disease management strategy in India.

The lignin riddle in jute: A comparative genomic investigation identifies targets for improving fiber quality

Jute fiber has become more popular in recent years due to its renewability, biodegradability, low manufacturing costs, and wide range of uses. However, one of the major components of fiber is lignin, which impairs the fiber quality and, as a result, makes jute fiber unsuitable for use in the textile industry and biofuel production. The availability of the jute (Corchorus olitorius) genome sequence provides the opportunity to perform a systematic analysis of the gene families and transcription factors specific to the lignin biosynthesis. Here, genome-wide screening was performed to identify the members of gene families associated with lignin biosynthesis in jute. Following this, phylogenetic analysis was carried out with functionally characterized genes involved in lignin biosynthesis of other species. The expression analysis among different tissues and different regions of bark tissue, taking into account fiber developmental stages, is also conducted to identify the genes mainly involved in jute fiber lignification. Fourteen genes and eleven transcription factors belonged to bona fide clades and showed strong expression in highly lignified tissues. Moreover, the transcript levels of these proteins were higher in the bottom bark tissues, which have a higher lignin accumulation compared to the top bark tissues, suggesting these genes were the core lignification genes in fiber formation. Similarly, gene ontology and protein-protein interactions analysis indicated that these genes were mainly involved in lignin biosynthesis and interacted with each other. The identification of the major lignification genes and transcription factors involved in jute fiber development provides a strong foundation for the development of jute cultivars to improve fiber quality.

Morphology and composition of calcium oxalate monohydrate phytoliths in the bark of Betula ermanii (stone birch): Case study from Sakhalin Island.

The morphology of calcium oxalate monohydrate precipitates (COM, Ca(C2O4)·H2O, P21/c, whewellite) occurring as crystals or intergrowths, as well as distribution of crystal-bearing idioblasts, have been studied for the first time in the bark of stone birch Betula ermanii from Sakhalin Island sampled in an area affected by mud volcanism and an unaffected typical forest environment taken for reference. The study addresses several issues (i) number and size of phytoliths and their distribution in different cell types; (ii) density of calcification in specific cells; (iii) habits of single crystals, twins, and complex intergrowths, as well as frequency of different morphologies and their relations. The trends of time-dependent morphological changes in separately analyzed crystals and intergrowths record the evolution of COM morphology from nuclei to mature grains. Of special interest are the nucleation sites and features of organic and inorganic seeds and nuclei for COM phytoliths. The precipitation process and crystal habits are mainly controlled by supersaturation, and it is thus important to constrain the Ca distribution patterns in different bark tissues. The B. ermanii samples were analyzed by several methods: scanning electron microscopy (SEM) for the distribution patterns and micromorphology of COM precipitates and bulk Ca content in bark; electron probe microanalysis (EPMA) for the mineral chemistry of COM precipitates; inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS) for trace elements in bulk bark and wood. RESEARCH HIGHLIGHTS: The distribution and morphology of whewellite precipitates in the analyzed B. ermanii bark samples indicate that the aqueous solution was most strongly supersaturated with respect to the Ca(C2O4)·H2O solid phase at the parenchyma-sclerenchyma boundary, where most of the COM spherulites are localized and often coexist with large single crystals and contact COM twins.

Wavy grain in sycamore maple (Acer pseudoplatanus) — a structural analysis of xylem and phloem

Summary Wavy grain (fiddle back) in trees has attracted the interest of both science and industry due to its aesthetic and economic importance. As the underlying causes of this particular structural feature in trees remain uncertain, this study investigates the possibility of non-invasively detecting wavy grain patterns within tree bark tissue of sycamore maple (Acer pseudoplatanus). Wavy grain, characterized by alternating light and dark stripes on the wood surface, is highly valued in the production of musical instruments. This research explores the potential of employing transmission light microscopy and microcomputer tomography (μCT) to examine the internal structure of phloem tissue to inspect whether the wavy grain structure found in the xylem continues in the bark. The investigation of phloem tissue proved challenging due to its irregular cellular arrangement. Nonetheless, slight phloem fibre deviations were observed in individual trees. However, no definite connection was found between the periodic oscillations of the xylem fibre tissue and the structure of the phloem in a given area. Variability in wavy grain expression among the xylem of the trees posed an additional challenge, with amplitudes and wavelengths differing across specimens. The application of μCT offered advantages over traditional sample preparation, enabling the study of the anatomical structure across larger areas and multiple depths without altering the specimen. This study sheds light on the potential of employing advanced imaging techniques to detect wavy grain patterns, providing insights that could benefit industries reliant on high-quality wood, such as musical instrument crafting and forestry.

Dynamics of 'Candidatus Liberibacter asiaticus' Growth, Concentrations of Reactive Oxygen Species, and Ion Leakage in Huanglongbing-Positive Sweet Orange.

Citrus Huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves-late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

The timing of wood formation in peatland trees as obtained with different approaches

Dendrometers offer valuable insights into how tree growth responds to climatic variables and physiological processes over the course of a year. Yet, their applicability to extremely slow-growing trees, such as those in peatlands, has been limited due to the intricate and slow nature of growth, therefore rendering interpretation of results complex. In this study, we conducted a comprehensive monitoring of tree wood formation in both peatland and mineral soil ecosystems in southern Sweden (58.37 N, 12.17 E, 75 m asl) in 2021 and 2022, using both band and point dendrometers. To verify and validate the dendrometer data, we also sampled microcores every two weeks during both growing seasons. We find that peatland trees grow at approximately 30 % the rate of their neighbors on mineral soils. The onset of growth among peatland trees typically occurs between mid-May and early June, consistently lagging the start of the growing season in trees on mineral soils by one to three weeks. Notably, growth peaks are synchronized across peatland trees and coincide with the summer solstice. Both types of dendrometers exhibit varying degrees of accuracy depending on the phenological stages measured. They perform well in identifying growth onset and peak but are less effective at detecting growth cessation. Point dendrometers demonstrate superior accuracy as they better capture daily irreversible growth increments. In the case of band dendrometers, growth increments are obscured by greater reversible fluctuations in dead bark tissues. However, they remain valuable for tracking the wood phenology of trees with growth rates exceeding 2 mm/year. Based on our results, for an effective tree monitoring in peatlands, we strongly recommend (1) using point dendrometers and (2) removing the dead bark tissues on monitored trees.

Effects of cold stress on protein metabolism of certain walnut cultivars

To investigate the effects of heat shock proteins (HSPs) on walnut (Juglans regia L.) plants under low-temperature stress, first of all, low-temperature tolerances of ten walnut cultivars (Chandler, Fernor, Franquette, Pedro, Bilecik, Kaman-I, Kaman-II, Kaman-III, Sebin, and Yalova I) were determined. One-year-old shoot samples were taken from the plants in two different periods, cold-acclimated (CA) and non-acclimated (NA), and were exposed to +5°C, -5°C, -15°C and -25°C for 12 h. Cold injury was determined by ion leakage analysis in the thawed bark tissues. According to the results of this analysis, two cultivars were determined as cold-sensitive (Chandler) and cold-tolerant (Fernor) in terms of low-temperature tolerance. To examine the effects of HSPs on cold tolerance in walnut plants, the protein profiles, the amount of total protein, and the HSPs of these two cultivars were determined. As a result of the immunoblot analysis, it was determined that 44 kDa HSP23 and 59 kDa HSP60 are responsible for low-temperature tolerance in walnut plants.

Identification of a cinnamoyl-CoA reductase from Cinnamomum cassia involved in trans-cinnamaldehyde biosynthesis.

The identification of a functional cinnamoyl-CoA reductase enzyme from Cinnamomum cassia involved in trans-cinnamaldehyde biosynthesis offers the potential for enhancing trans-cinnamaldehyde production through genetic engineering. A significant accumulation of trans-cinnamaldehyde has been found in the bark tissues of C. cassia, used in traditional Chinese medicine. trans-Cinnamaldehyde exhibits various pharmacological properties such as anti-inflammatory, analgesic, and protection of the stomach and the digestive tract. However, further elucidation and characterization of the biosynthetic pathway for trans-cinnamaldehyde is required. In this study, we conducted an integrated analysis of trans-cinnamaldehyde accumulation profiles and transcriptomic data from five different C. cassia tissues to identify the genes involved in its biosynthesis. The transcriptome data we obtained included nearly all genes associated with the trans-cinnamaldehyde pathway, with the majority demonstrating high abundance in branch barks and trunk barks. We successfully cloned four C. cassia cinnamoyl-CoA reductases (CcCCRs), a key gene in trans-cinnamaldehyde biosynthesis. We found that the recombinant CcCCR1 protein was the only one that more efficiently converted cinnamoyl-CoA into trans-cinnamaldehyde. CcCCR1 exhibited approximately 14.7-fold higher catalytic efficiency (kcat/Km) compared to the Arabidopsis thaliana cinnamoyl-CoA reductase 1 (AtCCR1); therefore, it can be utilized for engineering higher trans-cinnamaldehyde production as previously reported. Molecular docking studies and mutagenesis experiments also validated the superior catalytic activity of CcCCR1 compared to AtCCR1. These findings provide valuable insights for the functional characterization of enzyme-coding genes and hold potential for future engineering of trans-cinnamaldehyde biosynthetic pathways.

Phytophthora nicotianae and Ph. mediterranea: A biosecurity threat to Platanus orientalis and P. x acerifolia in urban green areas in Greece

Field surveys conducted in two urban green spaces in Athens (Greece) revealed the presence of Phytophthora-related diseases on Platanus orientalis and P. x acerifolia trees. Declining trees showed a range of disease symptoms including root rot, stem bleeding cankers and extensive canopy dieback. Since there is little information about the etiology of these diseases, a study was conducted from 2016 to 2021 to define the main pathogens involved. A total of 71 Phytophthora isolates were obtained from bark tissues and rhizosphere samples collected from symptomatic plane trees. Based on morphological traits and DNA sequence data, Phytophthora isolates were identified as Ph. mediterranea (11 isolates) and Ph. nicotianae (60). Over a five-year period 41% of P. orientalis trees associated with Phytophthora infections died and were removed from the park. Pathogenicity tests confirmed that all Phytophthora species including Ph. cinnamomi used for comparison, are pathogenic on both P. orientalis and P. x acerifolia trees. Phytophthora cinnamomi was shown to be the most aggressive species on both P. orientalis and P. x acerifolia. Phytophthora nicotianae was the dominant species, whereas Ph. mediterranea was associated with a few trees. Our finding has contributed to expanding knowledge on the host and geographic range of Ph. mediterranea, an invasive pathogen with a high potential for diffusion in Mediterranean regions and highlights the importance of enhancing biosecurity measures to prevent and limit the spread of invasive pathogens in urban and natural ecosystems.

Methods for the Determination of Fire Blight on Pome Fruit Crop Plants in the Western Part of Azerbaijan

Fire blight caused by Erwinia amylovora (Burrill, 1882) Winslow et al., 1920 causes significant production losses in the world’s leading pome fruit crop producing countries. The main route of infection of many fruit crops, including apple and pear trees, is through newly blooming flowers. The bacterium overwinters in the bark tissue along the edges of cankers formed on branches and stems as a result of infection in the previous year and becomes a source of infection in the next year. Determining the prevalence of the disease in areas with a large number of orchards is important from the point of view of disease control. Therefore, the purpose of the work is to study the prevalence of the disease in the Ganja-Dashkesan and Gazakh-Tovuz Economic Regions, as well as the accurate diagnosis of the obtained E. amylovora isolates using classical and molecular methods. For this purpose, trees in the studied apple and pear orchards were examined for the disease, the pathogen was isolated from infected plants, and morphological, biochemical, physiological and molecular analyzes were carried out. Among 36 bacterial isolates obtained from 27 samples of infected plants, Erwinia amylovora was identified in 11 as a result of morphological, biochemical, physiological and molecular tests.

Improved Canker Processing and Viability Droplet Digital PCR Allow Detection of Erwinia amylovora Viable Nonculturable Cells in Apple Bark.

The bacterium Erwinia amylovora causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen cells in fire blight cankers. Several factors may prevent E. amylovora from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 102 to 7.6 × 107 cells mL-1, as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of E. amylovora viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the E. amylovora life cycle, and elucidate E. amylovora physiology, epidemiology, and new options for canker management.

Endophytic fungi: isolated from Cosmos caudatus Kunth and Cosmos sulphureus Cav.: a histologic observation, identification, and secondary metabolites chemical analysis

Cosmos caudatus and Cosmos sulphureus are sorts of plant commonly use for side dish vegetables. Some plant live in mutualism symbiotic interaction with some endophytic fungi that protect the host plant. This research is done to: 1) determine the endophytic fungi colonization in the Cosmos plant tissues by histologic observation, 2) isolation and identify the endophytic fungi species from Cosmos leaf, branch bark and flower petal tissues, 3) analyze alkaloid, flavonoid, tannin, saponin, and phenolic contents produced by the each endophytic fungi species. The endophytic fungi culture on PDA medium were cut into 5×1 cm in size and inoculated in PDB medium, then shake in the rate of 120 rpm for 7×24 hours. Afterwards the liquid centrifugated. The secondary metabolites contents were analyzed by using spectrophotometric method. This research result showed that: 1) the endophytic fungi position was found on the leaf palisade cell wall, epidermal cell wall, sponsa cell wall, neighbour cell wall; on the branch bark parenchyme cell wall; on the flower petal epidermis cell wall; 2) eleven endophytic fungi have been identified; 3) some variation in the secondary metabolites contents produced by each endophytic fungi.

Bark structure is coordinated with xylem hydraulic properties in branches of five Cupressaceae species.

The properties of bark and xylem contribute to tree growth and survival under drought and other types of stress conditions. However, little is known about the functional coordination of the xylem and bark despite the influence of selection on both structures in response to drought. To this end, we examined relationships between proportions of bark components (i.e. thicknesses of tissues outside the vascular cambium) and xylem transport properties in juvenile branches of five Cupressaceae species, focusing on transport efficiency and safety from hydraulic failure via drought-induced embolism. Both xylem efficiency and safety were correlated with multiple bark traits, suggesting that xylem transport and bark properties are coordinated. Specifically, xylem transport efficiency was greater in species with thicker secondary phloem, greater phloem-to-xylem thickness ratio and phloem-to-xylem cell number ratio. In contrast, species with thicker bark, living cortex and dead bark tissues were more resistant to embolism. Thicker phellem layers were associated with lower embolism resistance. Results of this study point to an important connection between xylem transport efficiency and phloem characteristics, which are shaped by the activity of vascular cambium. The link between bark and embolism resistance affirms the importance of both tissues to drought tolerance.

Expanding the diversity of conifer xyloflora from Early Cretaceous Crato Fossil Lagerstätte, Brazil

Remains of gymnosperms are the most abundant plant macro-fossils in the outcrops of a fossil lagerstätte preserved in lacustrine limestones in the likely lower Aptian Crato Formation of the Araripe Basin in NE Brazil. However, the systematics of most fossil-taxa are mainly based on foliage and reproductive structures, whereas wood anatomy has rarely been used to study fossil plant diversity, despite the abundance of wood fragments. Here we describe a new fossil gymnosperm taxon based on anatomical characters of a well-preserved specimen from this lithostratigraphic unit. The new fossil-genus and fossil-species Cratoxylon placidoi gen. nov. et sp. nov. are established using characters of the secondary xylem and the bark, features that are also crucial for their affiliation into conifers. This finding reinforces the already known remarkable preservation potential of organisms in this konservat lagerstätte, since bark tissues are rarely preserved in the fossil record of Mesozoic gymnosperms. The results reveal the great potential of fossil woods for studying diversity of the Crato Formation flora and thus contribute to expand our knowledge on fossil vegetation in equatorial Gondwana during the early diversification of angiosperms.