Bark Tissues Research Articles

Plant life history strategies vary in subtropical forests with different disturbance histories: an assessment of biodiversity, biomass, and functional traits.

Disturbance alters environmental conditions in forests. Plants growing in forests with different disturbance histories in diverse environments may adopt varying life history strategies, but few studies focus on this effect. This study comprehensively investigated plant biodiversity, biomass, and functional traits in subtropical forests with two different disturbance histories in east China to explore differences in life history strategies. Biodiversity was slightly higher in disturbed compared to conserved forests. Significantly higher biomass was measured in conserved relative to disturbed evergreen broadleaved forests (P < 0.05). In conserved forests, leaf tissue density (LTD) was significantly higher and leaf thickness (LT), leaf dry matter content (LDMC), twig tissue density (TTD), twig dry matter content (TDMC), bark tissue density (BTD) and dry matter content (BDMC), and stem tissue density (STD) and dry matter content (SDMC) were significantly lower than in disturbed forests (P < 0.05). In terms of associated plant biodiversity, biomass, and functional traits, conserved forests adopted a resource acquisition strategy, reducing biodiversity and developing multiple functional traits such as high leaf area and specific leaf area and low LT, LDMC, TTD, TDMC, BTD, BDMC, STD, and SDMC to support a high biomass accumulation rate. Disturbed forests adopted a resource conservation strategy, enhancing biodiversity and developing converse trait combinations to lower the rate of biomass accumulation. A comprehensive investigation of plant biodiversity, biomass, and functional traits and subsequent assessment of plant life history strategies in conserved and disturbed forests will aid investigations of regional biodiversity and carbon reserves, contribute data to the TRY and Chinese plant trait databases, and improve ecological management and restoration efforts in east China.

Involvement of Phytophthora citrophthora and Phytophthora nicotianae in severe decline and gummosis of grapefruit (Citrus x paradisi Macfad.) in southern Türkiye

AbstractSince the summer of 2020, severe decline and gummosis were observed on 20‐ to 30‐year‐old grapefruit trees cv. Star Ruby in Antalya province in the Western Mediterranean region of Türkiye. Grapefruit trees were examined in affected commercial orchards in Aksu, Manavgat, Muratpaşa and Serik counties, located near the coast of the Mediterranean Sea. Symptomatic trees exhibited leaf discoloration, defoliation and severe dieback, as well as dark, water‐soaked lesions with gum exudates, characteristic of Phytophthora infections, formed on the bark of the main stem and major limbs. No Phytophthora species could be isolated from these bark tissues, while two were obtained from soil samples taken from around symptomatic trees. Based on morphological characteristics, isolates were identified as P. citrophthora and P. nicotianae. Identifications were confirmed by sequencing of the internal transcribed spacer (ITS) and the cytochrome oxidase I (cox1) gene regions. Pathogenicity tests, carried out by inoculations of the main stems and branches of grapefruit seedlings cv. Star Ruby indicated that both species were pathogenic. All P. citrophthora and P. nicotianae isolates obtained in this work were sensitive to mefenoxam. EC50 values of P. citrophthora isolates were between 0.284 and 0.285 μg mL−1, whereas those of P. nicotianae were between 0.288 and 0.291 μg mL−1. Phytophthora citrophthora was previously known to cause disease on citrus in Türkiye, but this is the first report of P. nicotianae causing citrus gummosis in the country. Because P. nicotianae is more widespread than P. citrophthora, it should be considered a threat to citrus cultivation in this region of Türkiye.

Modified Protocol for Isolation of High Quality RNA from the Matured Bark Tissue of tossa Jute

Modified Protocol for Isolation of High Quality RNA from the Matured Bark Tissue of tossa Jute

Isolation and in silico analysis of a transcript variant for a putative terpene synthase (HbTPS6L-X1) from the bark tissues of rubber Hevea brasiliensis RRIV 209

Hevea brasiliensis is the only tree species grown to produce natural rubber. The method of harvesting latex by slicing a groove into the bark of the tree mimics a mechanical wounding. In addition, rubber trees could be frequently exposed to pest and pathogen attacks through the scraped bark region. Terpenoids are a group of specialised compounds that play an important role in plant-environment interactions, especially in plant response to stress factors. The diversity of terpenoids is mainly determined by enzymes of the terpene synthase (TPS) family. Screening the RNA-seq data of H. brasiliensis revealed the presence of many putative TPS genes expressed in the bark tissues of rubber trees; however, their function has not been studied. This paper presents the identification of a TPS gene transcript variant (designated as HbTPS6L-X1) from the bark tissues of H. brasiliensis RRIV 209. Through the analysis of the phylogenetic tree, the presence of conserved motifs, and the signal peptide sequence region of the protein, as well as structural homology to known sesquiterpene synthases from other plants, HbTPS6L-X1 was predicted to belong to the TPS-a subfamily, be localised in the cytoplasm and catalyse the conversion of farnesyl diphosphate (FPP) to sesquiterpenes. These in silico functional characteristics are the basis for designing experiments to confirm the in planta biological function of HbTPS6L-X1 protein.

First Report of Root Rot Caused by Phytophthora lacustris on alder (Alnus lusitanica) in Spain.

Alder decline caused by Phytophthora species is considered one of Europe´s most important diseases of natural ecosystems. In Spain, P. x alni, P. uniformis, P. x multiformis and P. plurivora have been detected in association with root and collar rot in riparian alder populations (Pintos et al. 2010, 2012, 2017; Haque et al. 2014). During the active growth periods 2020-2021, a field survey of the newly designed species Alnus lusitanica (Vít et al. 2017) was carried out at the most symptomatic areas along the Miño-Sil river basin in Galicia and León (NW-Spain). Samples of bark, root, rizosphere soil of declining trees, and river water were collected. Symptoms, similar to those caused by other Phytophthora species, included low leaf density of the canopy, generally with smaller and chlorotic leaves, and branches with dry tips. In some cases, the presence of cankers on the trunk were observed (Figure 1). Necrotic lesions were transferred onto V8-PARPH, a Phytophthora selective medium. Soil and water were baited with carnation petals. Three isolates of one Phytophthora species was recovered: two from the roots of trees, and one from river water at three different sites. The isolates were transferred to V8 agar and incubated a 22ºC in the dark. Colonies had petaloid patterns, and their optimum growth temperature ranged from 25 to 30 °C. In soil extract, non-caducous, non-papillate and ovoid sporangia were produced. Sporangia averaged 41.0×29.4 μm with a length/breadth ratio of 1.39. Internal proliferation occurred (Figure 2). No sexual structures were observed . Genomic DNA was extracted from mycelium obtained from pure cultures of three Phytophthora isolates. The ITS region of the ribosomal DNA template was performed by nested PCR using DC6 (Cooke et al. 2000) and ITS4 (White et al. 1990) in the first round, and ITS6 (Cooke et al. 2000) and ITS4 in the second. The mitochondrial gene cox1 was amplified with primers CoxF4N and CoxR4N (Kroon et al. 2004). BLASTn analyses showed 100% identity to the type of P. lacustris (AF266793, 156 matching bp) for ITS, and 100% identity to the ex-type of P. lacustris (MH136916, 596 matching bp) for the cox1 sequence. Phylogenetic analysis indicated that our isolates were localized in the same evolutionary branch as P. lacustris based on consense sequences of ITS and cox1 sequences. The ITS and cox1 sequences generated in this study were deposited in GenBank with accession number OP588369 and OP548051, and one isolate isolated from root (CECT 21212) was submitted to the Spanish Type Culture Collection (Paterna, Valencia). Pathogenicity tests with isolate CECT 21212 were conducted on ten 3-year-old alders (A. lusitanica) growing in free draining 5 L pots. One shallow cut was made into the cambium at the root collar level. A colonized 5-mm mycelial agar plug, from a 7-day-old culture was inserted into every wound (mycelial surface face-down) and sealed with Parafilm®. Five control plants were inoculated with a sterile agar plug. Plants were kept in a controlled chamber at 25 ºC and 80% humidity. After a 3-week incubation period, inoculated plants showed dieback symptoms and necrosis of the inner bark tissue (Figure 3). Lesion lengths ranged from 2 to 8 cm. Control plants remained symptomless. P. lacustris was recovered from all inoculated plants, but not from the control ones. To our knowledge, this is the first report of P. lacustris causing root rot on alders in Spain. This new detection represents an increased threat to riparian alders by the presence of an additional Phytophthora species associated with alder decline, since P. lacustris can readily adapt to a wide variety of climatic conditions with the ability to infect different hosts (Nechwatal et al. 2013).

Evaluation of Natural Spring Freeze Tolerance of Five Pecan Scion–Rootstock Combinations

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is a member of the Juglandaceae family. During spring, pecan trees break their bud dormancy and produce new leaves and flowers. Carbohydrates stored in roots and shoots are thought to support the bloom and early vegetative growth during this time until new leaves start the full photosynthetic activity. Spring freeze is known for its damaging effects on pecan bud and flower growth and development. Pecan shoots with leaves and flowers from five scion–rootstock combinations were collected hours before and after a recent spring freeze (below 0 °C for 6 hours, 21 Apr 2021, Perkins, OK, USA). Morphologies of the leaf, bud, and catkin were visually observed, and the morphologies of the anther and pollen in paraffin sections were investigated by light microscopy. Soluble sugar and starch from bark and wood were analyzed using the anthrone reagent method. The Kanza–Mount showed the maximum damage to terminal leaves, buds, and catkins, whereas Maramec–Colby had the minimum damage only to leaves. Pollen grains were shrunk and reduced in number in the anthers in the protandrous Pawnee scions, whereas no pollen damage was observed in the protogynous Kanza scion. Furthermore, bark soluble sugar levels increased in all the scion–rootstock combinations after the freeze, which may indicate a physiological response to the cold stress. Overall, the extent of spring freeze damage of pecans is affected by the growth stage, types of scion and rootstock, and the scion–rootstock interactions. Furthermore, in addition to low temperature, scion–rootstock interactions also affected the starch and soluble sugar contents in wood and bark tissues.

Signatures of prescribed fire in the microbial communities of Cornus florida are largely undetectable five months post-fire.

Prescribed burn is a management tool that influences the physical structure and composition of forest plant communities and their associated microorganisms. Plant-associated microorganisms aid in host plant disease tolerance and increase nutrient availability. The effects of prescribed burn on microorganisms associated with native ecologically and economically important tree species, such as Cornus florida L. (flowering dogwood), are not well understood, particularly in aboveground plant tissues (e.g., leaf, stem, and bark tissues). The objective of this study was to use 16S rRNA gene and ITS2 region sequencing to evaluate changes in bacterial and fungal communities of five different flowering dogwood-associated niches (soil, roots, bark, stem, and leaves) five months following a prescribed burn treatment. The alpha- and beta-diversity of root bacterial/archaeal communities differed significantly between prescribed burn and unburned control-treated trees. In these bacterial/archaeal root communities, we also detected a significantly higher relative abundance of sequences identified as Acidothermaceae, a family of thermophilic bacteria. No significant differences were detected between prescribed burn-treated and unburned control trees in bulk soils or bark, stem, or leaf tissues. The findings of our study suggest that prescribed burn does not significantly alter the aboveground plant-associated microbial communities of flowering dogwood trees five months following the prescribed burn application. Further studies are required to better understand the short- and long-term effects of prescribed burns on the microbial communities of forest trees.

First Report of Xanthomonas arboricola pv. corylina Causing Bacterial Blight on Hazelnut Tree (Corylus avellana) in Montenegro.

Hazelnut is a minor but rapidly increasing commercially grown species in Montenegro. In June 2021, severe infection, affecting more than 80% of the trees, was observed on 6-year-old hazelnut plants (Corylus avellana) cultivar Hall's Giant, in a 0.3ha plantation near Cetinje, central Montenegro. Numerous, small, 2-3mm in diameter, irregular, brown, necrotic spots, sometimes surrounded by a weak chlorotic halo, were observed on leaves. As the disease progressed, the lesions coalesced and formed large necrotic areas. Necrotic leaves remained attached to the twigs. Longitudinal brown lesions developed on twigs and branches, causing their dieback. Necrotic, unopened buds were noticed as well. No fruits were observed in the orchard. From the diseased leaf, bud and twig bark tissue, yellow, convex, and mucoid bacterial colonies were isolated on yeast extract dextrose CaCO3 medium and 14 isolates were subcultured. The isolates induced hypersensitive reaction in pelargonium leaves (Pelargonium zonale), were Gram-negative, catalase positive, oxidase negative, obligate aerobic, hydrolyzed starch, gelatin and esculin, did not reduce nitrate and did not grow at 37°C and in the presence of 5% NaCl, showing so the same biochemical profile of the reference strain Xanthomonas arboricola pv. corylina (Xac) NCPPB 3037. Using primer pair XarbQ-F/XarbQ-R (Pothier et al., 2011), a 402 bp product was amplified in all 14 isolates and the reference strain, confirming their affiliation to X. arboricola species. Additionally, the isolates were further identified by PCR analysis, using primer pair XapY17-F/XapY17-R (Pagani 2004; Pothier et al., 2011), resulting in a single band of 943 bp characteristic for Xac. The amplification and sequencing of the partial rpoD gene sequence of two selected isolates RKFB 1375 and RKFB 1370, were performed using a set of primers described by Hajri et al., 2012. The obtained DNA sequences showed that the isolates (GenBank Nos. OQ271224 and OQ271225) share 99.47% to 99.92% rpoD sequence identity with Xac strains CP076619.1 and HG992342.1 isolated from hazelnut in France and HG992341.1 in USA. Pathogenicity of all isolates was confirmed by spraying young shoots (20 to 30 cm long, with 5 to 7 leaves) on 2-year-old potted hazelnut plants (cv. Hall's Giant) using a handheld sprayer with the bacterial suspension (108 CFU/mL of sterile tap water), in three replicates. Sterile distilled water (SDW) and NCPPB 3037 Xac strain were used as negative and positive control, respectively. The inoculated shoots were incubated under plastic bags, providing high humidity conditions, in an acclimatized greenhouse at 22-26°C, for 72 h. Lesions surrounded by a halo appeared on leaves of all inoculated shoots within 5 to 6 weeks after inoculation, while leaves sprayed with SDW remained symptomless. Koch's postulates were confirmed by the re-isolation of the pathogen from the necrotic test plant tissue and identity checked by PCR using the primer set of Pothier et al., 2011. Based on pathogenic, biochemical, and molecular characteristics, the isolates from hazelnut plants in Montenegro were identified as X. arboricola pv. corylina. This is the first report of Xac affecting hazelnut in this country. Considering favorable environmental conditions, the pathogen can cause significant economic losses in hazelnut production in Montenegro. Therefore, phytosanitary measures have to be implemented to prevent introduction and spread of the pathogen in other areas.

A review of low-frequency latex harvesting systems that lessen the tapper shortage problem of the smallholders' natural rubber production

Smallholders' rubber production is encountering problems of skilled tapper shortage and high production costs resulting from increased worker wages and the substantial growth of new mature areas. Low-frequency latex harvesting system (LFLHS) effectively improves tapper productivity with long-term optimum yield by reducing the tapper requirement. LFLHS reduces tapper requirement by 33% to 67% of the conventional harvesting systems. Under the d3 (tapping every three days) frequency harvesting system, a tapper is assigned to cover three tasks, and his productivity is at least 30% higher than that of the d2 (alternative daily) frequency harvesting system. The cumulative yield of LFLHS is comparable to that of d2 frequency. It is economically profitable when the cumulative yield of LFLHS reaches 90% of the d2 frequency tapping as a break-even yield. Its low number of tapper requirement and high productivity saves tapping cost. 20% to 55% of tapping cost can be reduced by shifting the harvesting frequency from d2 to d3. The virgin bark of basal panels could be tapped at least four to ten years more than conventional tapping systems. The low bark consumption allows sufficient time for the regeneration of bark tissues resulting in a potentially higher yield from the renewed bark. Thus, sustainable economic yield is achievable for a productive lifespan of 30 to 35 years from the LFLHS. These advantages of LFLHS contribute to reducing the tapper requirement and cost of production, ensuring increased profits and a longer economic lifespan of rubber production.Keywords: Latex harvesting system, Tapper requirement, Tapper productivity, Tapping cost

First Report of Stem Rot Caused by Botryosphaeria dothidea and Phomopsis velata on Forsythia suspensa in China.

Forsythia suspensa (Thunb.) Vahl (Oleaceae) is a well-known traditional Chinese medicine. It exhibits antioxidant activity and exerts antibacterial, antiviral, and antiemetic effects (Li and Chen 2005). From May 2020 to October 2021, a disease was observed on field-grown forsythia plants in Lingbao City, Henan Province, China (110°33'25.74″E, 34°30'19.34″W). The diseased plants were characterized by stem rot, retarded growth, a declined fruit quality, and in extreme cases, death of F. suspensa. Approximately 3.0% to 5.0% individuals exhibited stem rotten in the main branches. On average, 60% of the branches of infected individual trees were affected by this disease. During the initial infection stage, the bark of the plants was raised and curled, and the xylem and phloem of the stems turned brown color, whereas in the late stage of the infection, the outer bark had dried and become detached, and the inner xylem and phloem had blackened. Upon infection, the growth of plants was reduced, and the main branches became desiccated as the disease progressed. We randomly selected five diseased branches from five plant fields, the bark tissues (about 25 mm²) of which were surface-sterilized in 75% ethanol for 30 s, treated with 1% NaClO for 5 min, rinsed five times with sterile water, and placed on potato dextrose agar (PDA). After incubating 3 days, 20 clones were observed, and two representative strains (FSJF11 and FSJF13, three replicates for each) was selected for intensive study. Samples of these strains have been deposited in Institutes of Traditional Chinese Medicine, Henan University. On PDA, the colonies of FSJF11 were initially white and fluffy in appearance, later turning gray, and finally black. The vigorously growing hyphae were branched and septate. However, no spores was observed during culture. FSJF13 colonies were rapidly growing, initially white in color and later turning gray. After culturing for 20 days, black conidia appeared and yellow conidial horns were released. The alpha conidia were elliptical, slightly pointed at both ends, and each end possessed an oil ball (6.40±0.60 × 1.86±0.25 μm). The beta conidia were slender, linear, and hook shaped with a slightly curved end (28.92±2.81 × 0.96±0.14 μm). DNA of the isolates was extracted using a Fungal Genome DNA Extraction Kit (Sangon Biotech, Shanghai), and selected genes were amplified using the primer pairs ITS1/ITS4 (Tian et al. 2018), LROR/ LR5, and NS1/NS4 (Aiello et al. 2020). Sequences have been deposited in GenBank (ITS: MW834579 and MW834580; LSU: MW829566 and MW829567; SSU: MW834582 and MW834583). The lengths of the amplified ITS, LSU and SSU sequences were 491, 759, and 1013 bp for FSJF11, respectively, and these in FSJF13 were 543, 927, and 901 bp, respectively. The ITS, LSU, and SSU sequences of FSJF11 were found to have sequence identities of 99.19%, 100%, and 100% with those of Botryosphaeria dothidea stains AY259092, EU673243, and Eu673174, respectively, and a phylogenetic tree was constructed based on the concatenated sequences (ITS, LSU, and SSU) revealed that FSJF11 and B. dothidea formed a clade with 96% support. A BLAST search of the Genbank database revealed that the ITS sequence of FSJF13 showed 99.81% identity with that of Phomopsis velata (MN183778). Given that no LSU or SSU sequences of this species are currently available, we constructed a phylogenetic tree based solely on ITS sequences, which revealed that FSJF13 and P. velata formed a clade with 99% support. Based on the morphological and molecular characteristics(Qi et al. 2007), the isolates of FSJF11 and FSJF13 were identified as B. dothidea and P. velata, respectively. Healthy branches of F. suspensa were wounded in vitro after inoculating active fungal cake of B. dothidea or P. velata (diameter = 5 mm) on the bark, and control branches were treated with PDA. In total, each branch was inoculated via four holes were inoculated on each branch, and three branches were used for each treatment. The inoculation sites were covered with a piece of wet absorbent cotton and then wrapped with plastic film, and the branches were incubated at 26 °C. The branches continued to grow after removal of the cotton and the film on the fourth day. All inoculated points on the branches showed lesions similar to those observed in the field, whereas the control branches were asymptomatic. The pathogenicity rates of FSJF11 and FSJF13 (three replicates for each) were 66.67% and 83.33%, respectively. Both species were re-isolated from the symptomatic branches respectively, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of B. dothidea and P. velata causing branches rot in F. suspensa. The findings of this study will contribute to developing effective strategies for the control of this newly emerging plant disease.

A method to quantify and account for the hygroscopic effect in stem diameter variations

Dendrometers recording stem diameter variations (SDV) at high-resolution are useful to assess trees' water relation since water reserves are stored in the elastic tissue of the bark. These tissues typically shrink during the day as they release water when evaporative demand is high and swell during the night as they are replenished when evaporative demand is low, generating the typical SDV profile known as the diel SDV cycle. However, similar SDV cycles have been observed on dead trees due to the hygroscopic shrinking and swelling of the dead bark tissues. In order to remove this hygroscopic effect of the bark, dendrometers are applied as close as possible to the living bark tissues by removing the outer dead layer, however with questionable success. In this study, we used SDV time series from 40 point dendrometers applied on dead-bark-removed mature trees to assess and quantify the remaining hygroscopic effect on individual trees. To do so, we checked SDV behavior in the cold season and explored the relation between the diel SDV cycle and changes in relative humidity (RH). Our results showed that (a) the hygroscopic effect in SDV can be well-detected based on the amplitude of the diel SDV cycle (diel SDVampl) and the correlation between SDV and RH during both the cold and the warm season; (b) the level of the hygroscopic effect varies strongly among individuals; (c) diel SDVampl is proportional to both changes in RH and transpiration so that the hygroscopic effect on the diel SDV cycle can be quantified using a linear model where (diel SDVampl) is a function of RH changes and transpiration. These results allow the use of the model to correct the amplitude of the diel SDV cycles and suggest that this method can be applied to other ecological relevant water-related SDV variables such as tree water deficit.

Global characteristics and drivers of sodium and aluminum concentrations in freshly fallen plant litter.

Plant litter is not only the major component of terrestrial ecosystem net productivity, the decomposition of which is also an important process for the returns of elements, including sodium (Na) and aluminum (Al), which can be beneficial or toxic for plant growth. However, to date, the global characteristics and driving factors of Na and Al concentrations in freshly fallen litter still remain elusive. Here, we evaluated the concentrations and drivers of litter Na and Al with 491 observations extracted from 116 publications across the globe. Results showed that (1) the average concentrations of Na in leaf, branch, root, stem, bark, and reproductive tissue (flowers and fruits) litter were 0.989, 0.891, 1.820, 0.500, 1.390, and 0.500 g/kg, respectively, and the concentrations of Al in leaf, branch, and root were 0.424, 0.200 and 1.540 g/kg, respectively. (2) mycorrhizal association significantly affected litter Na and Al concentration. The highest concentration of Na was found in litter from trees associated with both arbuscular mycorrhizal fungi (AM) and ectomycorrhizal fungi (ECM), followed by litter from trees with AM and ECM. Lifeform, taxonomic, and leaf form had significant impacts on the concentration of Na and Al in plant litter of different tissues. (3) leaf litter Na concentration was mainly driven by mycorrhizal association, leaf form and soil phosphorus concentration, while leaf litter Al concentration was mainly controlled by mycorrhizal association, leaf form, and precipitation in the wettest month. Overall, our study clearly assessed the global patterns and influencing factors of litter Na and Al concentrations, which may help us to better understand their roles in the associated biogeochemical cycles in forest ecosystem.

Diversity and antioxidant activity of endophytic fungi isolated from salam (Syzygium polyanthum)

Abstract. Widjajanti H, Elfita, Sari MT, Hidayati N, Hariani PL, Setiawan A. 2023. Diversity and antioxidant activity of endophytic fungi isolated from salam (Syzygium polyanthum). Biodiversitas 24: 3051-3062. Salam (Syzygium polyanthum (Wight) Walp.) is a medicinal plant from the family Myrtaceae. The leaves are used as a spice and treat diseases such as diabetes mellitus, hypertension, ulcers, diarrhea, gastritis, and skin. This study aimed to determine endophytic fungi's diversity and antioxidant activity in the S. polyanthum plant. In addition, the distribution of endophytic fungi found on S. polyanthum was compared to that of previously reported endophytic fungi on other species of Syzygium. The distribution pattern of these endophytic fungi is needed to determine the presence of potential endophytic fungi that can live in host plants of the same genus. The endophytic fungi were isolated from fresh root bark, stem bark, and leaf tissues of S. polyanthum. The endophytic fungi were identified morphologically to determine their level of diversity; their extract was then tested for their antioxidant activity using the DPPH method. The extract that showed the highest antioxidant activity was identified molecularly. A total of 18 endophytic fungi were obtained from S. polyanthum, including seven isolates from root bark (HSA1–HSA7), four isolates from stem bark (HSB1–HSB4), and seven isolates from leaves (HSD1–HSD7). The morphological identification showed seven genera scattered in all investigated parts: Trichoderma, Penicillium, Aspergillus, Pythium, Papulaspora, Pythiogeton, and Clonostachys. The HSD5 isolate showed the best antioxidant activity, and the molecular identification confirmed this isolate as Clonostachys rosea. The comparison of the distribution of endophytic fungi isolated from S. polyanthum to endophytic fungi isolated from the host genus Syzygium showed that endophytic fungi of three genera, namely Clonostachys, Papulaspora, and Pythiogeton, are specifically found on S. polyanthum. In addition, those of four other genera, namely Trichoderma, Aspergillus, Penicillium, and Pythium, are found on other species of Syzygium.

Should more individuals be sampled when measuring functional traits of tree species in habitat-heterogeneous karst forests?

When measuring plant functional traits across geomorphologies, 5–10 healthy individuals of a plant species are commonly sampled. However, whether more individuals should be sampled in habitat-heterogeneous karst vegetation remains unknown. In this study, two dominant tree species (Clausena dunniana and Platycarya strobilacea) in karst evergreen and broadleaved mixed forests in Southwestern China were selected. On the basis of a large quantity of individuals of the two species grown in different peak clumps and slope positions, variations of 10 morphological traits in the two species were statistically analyzed. The suggested sampling number of individuals, which could mostly represent the common trait characteristics, was further explored. All traits showed significant differences between the two species (p &amp;lt; 0.05). The traits of P. strobilacea displayed larger intraspecific variations than those of C. dunniana, except for twig dry matter content. The bark thickness (BT), leaf area (LA), and specific leaf area (SLA) of C. dunniana and the BT, SLA, LA, leaf tissue density, and bark tissue density of P. strobilacea presented large intraspecific variations. Most traits exhibited significant differences between peak clumps and/or among slope positions (p &amp;lt; 0.05). Random sampling analysis indicated that the suggested sampling numbers of individuals for the 10 traits are 6–23 in C. dunniana and 9–29 in P. strobilacea. The common accepted sample size in normal geomorphologies is not sufficiently large in most cases. Larger sample sizes are recommended for traits, such as SLA, BT, and LA, with larger intraspecific variations. Therefore, under sufficient labor, material, and time, more individuals should be sampled when measuring plant functional traits in habitat-heterogeneous karst vegetation.

Endophytic fungi isolated from the root bark of sungkai (Peronema canescens) as Anti-bacterial and antioxidant

Traditional medicine is still used by the community to prevent and treat various diseases, including infectious diseases and exposure the free radicals. The part of the medicinal plant that is often used as traditional medicine is root bark of sungkai. This study explored endophytic fungi from root bark of sungkai and determined their antibacterial and antioxidant activities. Endophytic fungi were isolated from fresh root bark tissue in PDA (Potato Dextrose Agar) media and identified morphologically. The DPPH (2,2-diphenyl-1-picrylhydrazyl) and diffused-disc paper methods were used for antioxidant and antibacterial. The endophytic fungi extracts showing the best bioactivity were identified molecularly. A total of 6 isolates of endophytic fungi were found in this study (RNA1-RNA6). Antibacterial and antioxidant activity test showed the best activity for RNA4 isolates. Molecular identification results describe it as Penicillium janczewskii. This research can be used as a basic reference for isolating pure compounds that have antibacterial and antioxidant properties through chromatography techniques

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering.

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while "Candidatus Liberibacter asiaticus" has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California's citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step. Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/µL) that was comparable with the CCPP's hand-chopping protocol (77.84 ng/µL). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

Transcriptomic alterations in the sweet orange vasculature correlate with growth repression induced by a variant of citrus tristeza virus.

Citrus tristeza virus (CTV, family Closteroviridae) is an economically important pathogen of citrus. CTV resides in the phloem of the infected plants and induces a range of disease phenotypes, including stem pitting and quick decline as well as a number of other deleterious syndromes. To uncover the biological processes underlying the poorly understood damaging symptoms of CTV, we profiled the transcriptome of sweet orange (Citrus sinensis) phloem-rich bark tissues of non-infected, mock-inoculated trees and trees singly infected with two distinct variants of CTV, T36 or T68-1. The T36 and T68-1 variants accumulated in the infected plants at similar titers. With that, young trees infected with T68-1 were markedly repressed in growth, while the growth rate of the trees infected with T36 was comparable to the mock-inoculated trees. Only a small number of differentially expressed genes (DEGs) were identified in the nearly asymptomatic T36-infected trees, whereas almost fourfold the number of DEGs were identified with the growth-restricting T68-1 infection. DEGs were validated using quantitative reverse transcription-PCR. While T36 did not induce many noteworthy changes, T68-1 altered the expression of numerous host mRNAs encoding proteins within significant biological pathways, including immunity and stress response proteins, papain-like cysteine proteases (PLCPs), cell-wall modifying enzymes, vascular development proteins and others. The transcriptomic alterations in the T68-1-infected trees, in particular, the strong and persistent increase in the expression levels of PLCPs, appear to contribute to the observed stem growth repression. On the other hand, analysis of the viral small interfering RNAs revealed that the host RNA silencing-based response to the infection by T36 and that by T68-1 was comparable, and thus, the induction of this antiviral mechanism may not contribute to the difference in the observed symptoms. The DEGs identified in this study promote our understanding of the underlying mechanisms of the yet unexplained growth repression induced by severe CTV isolates in sweet orange trees.

Callose synthase and xyloglucan endotransglucosylase gene expression over time in Citrus × clementina and Citrus × sinensis infected with citrus tristeza virus

Citrus tristeza virus (CTV) is a virus that already caused great losses in citrus producing regions. The cell wall of plant cells plays an important role in the defence response to viruses. Following several studies indicating that cell wall enzyme transcripts of callose synthase 7 (calS7) and xyloglucan endotransglucosylase 9 (xth9) are modified during a viral infection, transcript expression of calS7 isoform x5 (calS7x5) and xth9 was evaluated over time in Citrus × sinensis ‘Valencia Late’ (VL) and Citrus × clementina ‘Fina’ (CL), infected with the severe CTV isolate T318A, by quantitative (q) PCR. qPCR analysis of healthy and CTV infected citrus was performed at 15 days, 10 months and at 31 months post-inoculation (dpi/mpi), respectively. The CTV titer, evaluated at the three time-points by qPCR, increased over time in bark tissues, with VL plants exhibiting a titer about 5 times higher than CL 31 mpi. CTV infection did not cause significant changes in calS7x5 gene expression over time in both citrus cultivars. However, CTV infection was associated with significant up-regulation of xth9 in VL compared to controls 31 mpi. This study highlights that CTV infection can affect the expression of specific cell wall-associated genes over time and that this influence was distinct for VL and CL. This study provides further insight into the CTV-citrus host interaction, with the long-term response of VL to a severe CTV isolate involving a high expression of the xth9 gene.

First report of Diaporthe eres causing stem blight and dieback on highbush blueberry (Vaccinium corymbosum) in Italy.

Vaccinium corymbosum L. is a woody deciduous shrub belonging to the Ericaceae family. During the last decade, Northern Italy has become a major area for blueberry cultivation and production in Europe, supplying other countries with high quality fruit (FAOSTAT 2020). In June 2020, plant stem and branch wilting with brownish necrotic internal lesions were observed on 20% of around 250 plants of the cultivar (cv.) Draper cultivated in a soilless culture system located in Peveragno (Piedmont, Cuneo Province, 44°21'05.6''N, 07°37'23.2''). Dieback and death of the plants also occurred. Fifteen symptomatic plants were collected. Wood pieces (5-10 mm) excised from diseased stems and branches were sterilized in 1% sodium hypochlorite for 1 min, rinsed in sterile distilled water and dried on sterile absorbent paper. Small fragments (2-3 mm), obtained from the edge of the necrotic tissues, were plated on potato dextrose agar (PDA) amended with 25 mg/L of streptomycin sulfate. Plates were incubated at 25 ± 1°C under a 12 h photoperiod and, five days after, colonies with the same characteristics of Diaporthe spp. were established from single hyphal tip transfers. Two representative strains (CVG1130 and CVG1131) were grown on PDA from single hyphal-tip transfers. After 7 days, white colonies with fluffy, aerial, mycelium reaching 8.3 cm were observed. colonies turned gray after three weeks producing dark brown pycnidia. Aseptate, hyaline, fusiform to ellipsoidal, measuring 6.3 to 8.4 × 2.2 to 3.0 μm alpha conidia were produced. No beta conidia were observed. The DNA of the same strains was extracted and a polymerase chain reaction (PCR) was performed for the ITS (primer set: ITS1-ITS4) (GenBank Accession no. ON834528; ON834529), tef (EF1-728/EF1-986) (GenBank Accession no. ON843715; ON843716) and tub2 (T1/Bt2b) (GenBank Accession no. ON843713; ON843714) regions, in accordance with previous studies (Gomes et al. 2013, Bezerra et al. 2021). Sequences analysed with BLASTn algorithm (Altschul et al. 1997) exhibited 98% identity with the ex-type strain CBS 138594 of Diaporthe eres for ITS (GenBank Accession no. KJ210529), 100% identity for tub2 (GenBank Accession no. KJ420799) and 99% identity for tef (GenBank Accession no. KJ210550). The maximum likelihood method based on combined sequences of ITS, tef and tub2 loci was performed, and the isolates CVG1130 and CVG1131 clustered with several reference strains of D. eres (Supplementary figure). To fulfil Koch's postulates, pathogenicity tests were performed on 1-year-old blueberry potted plants cv. Draper and Duke. A small piece of the bark tissue was removed with a sterile-bladed scalpel to expose the cambium. Mycelium plugs (5 mm diameter), obtained from 10-day-old cultures of the strains CVG1130 and CVG1131, were placed with the mycelium in contact with the internal plant tissues. Three plants were used for each isolate and the inoculation points were sealed with Parafilm®. The same number of plants treated with sterile PDA plugs were used as control. Plants were placed in a growth chamber at 25°C for 3 weeks. After this period, symptoms similar to those observed in the field appeared on the inoculated plants, while control plants remained healthy. A fungus with the same morphological characteristics of D. eres was reisolated from inoculated plants and identified by sequencing the tub2 gene to confirm Koch's postulates. Diaporthe eres was previously reported on different Vaccinium spp. in Chile, Lithuania, the Netherlands and the U.S.A. (Farr and Rossman, 2022). To our knowledge, this is the first report of D. eres associated with stem blight and dieback of highbush blueberry in Italy. Duke and Draper are among the most cultivated blueberry cultivars in Piedmont, where the spread of D. eres could represent a serious threat.

Genome-wide identification and expression analysis of MYB transcription factors involved in fiber formation in white jute (Corchorus capsularis)

MYB transcription factors (TFs) are vital players in various plant biochemical and physiological processes. To date, MYB TFs have been comprehensively investigated in numerous plant species. Corchorus capsularis is one of the most important fiber crops worldwide. However, systematic investigation of the MYB TFs in C. capsularis remains elusive, which limits our insights into the contribution of MYB TFs regulating numerous biochemical processes such as anthocyanin accumulation and physiological processes (Color, growth, etc.) in this crop. Therefore, this study systematically investigated MYB genes comprising the gene structure, phylogeny, chromosomal locations, protein motifs, expression patterns, protein-protein interaction, and collinearity analysis. A total of 88 Corchorus capsularis R2R3-MYB (CcMYB) genes were characterized and distributed on seven chromosomes. The collinearity study revealed that the segmental duplication events were responsible for the enlargement of CcMYB genes. The highest syntenic relationship was observed between CcMYB genes (70/88) and Gossypium hirsutism genes. Further, phylogenetic tree analysis classified the 88 CcMYBs into 25 subgroups based on domain similarity and phylogenetic topology. Furthermore, the phylogenetic investigation of CcMYBs and Arabidopsis MYBs displayed the functional discrepancy of the CcMYB genes throughout evolution. Changes in anthocyanin content were observed among white jute’s stem bark tissues, which may be due to their different colors. Expression study of 11 CcMYB genes in total by real-time quantitative PCR revealed the diverse expression profiles in various tissues organs and functional predictions found that some CcMYBs might be associated with stem bark color and anthocyanin content, cellulose, and lignin biosynthesis. Co-expression interaction analysis showed that CcMYB genes could be involved in physiological, biological, and molecular processes. Our study provides comprehensive details on MYB genes and contributes to a better understanding of their functions in Corchorus capsularis.