Maclura, a small genus in the Moraceae family, is widely distributed across East and Southeast Asia and is recognized for its considerable ecological, economic, medicinal, and social significance. In this study, we sequenced, assembled, and annotated the complete chloroplast genome of Maclura tricuspidata and conducted comparative analyses with three previously published chloroplast genome of the genus (M. tricuspidata MW732703.1, M. tricuspidata NC_056295.1, and M. cochinchinensis NC_066228.1). The chloroplast genome is 161,351 bp in length and displays the canonical quadripartite architecture, consisting of a large single-copy (LSC) region of 89,382 bp, a small single-copy (SSC) region of 20,231 bp, and a pair of inverted repeat (IR) regions, each measuring 25,869 bp. Genome annotation identified 112 distinct genes, including 78 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes, among which 18 genes are duplicated within the IR regions. Comparative boundary analyses revealed that several genes, including rpl2, rpl22, rps19, ndhF, ycf1, psbA and trnH, are positioned at or span the junctions between the LSC, SSC, and IR regions. Although the overall chloroplast structure is highly conserved among the four Maclura accessions, minor variations were detected in the lengths of the LSC, SSC, and IR regions. Phylogenomic analysis strongly supported the monophyly of Maclura within Moraceae, indicating close evolutionary relationships among the species. These findings provide valuable genomic resources for future phylogenetic and evolutionary research in Moraceae and enhance our understanding of chloroplast evolution within the genus Maclura.

Dry dipterocarp forests (DDFs) in Thailand are vital ecosystems that support the growth of edible mushrooms and other non-timber forest products (NTFPs). However, destructive burning practices to harvest NTFPs particularly Astraeus odoratus, a valued ectomycorrhizal (ECM) fungus, account for more than 50

The effect of ectomycorrhizal (ECM) fungi on the absorption and transport of heavy metals by host plants remains elusive. We experimentally assessed rapid cadmium (Cd) diffusion by two species of Suillus mycelium. Furthermore, we evaluated Cd absorption by ECM Pinus thunbergii Parl. and used transcriptomics to study the gene expression of P. thunbergii under Cd stress. In vitro experiments revealed that Cd2+ was transported through the apoplastic space more rapidly than through the mycelial symplast. The net Cd2+ influx rates in epitaxial hyphae were the highest, followed by those in the mantle of P. thunbergii inoculated with Suillus, whereas the lowest influx rate was found in the ECM-free fine root portions. Under Cd stress, the expression levels of PtZnTs, PtZIPs and PtHMA2 in ECM P. thunbergii roots were significantly higher than those in non-mycorrhized P. thunbergii. The assessment of Cd distribution in P. thunbergii revealed that Cd was transported to the needles of ECM P. thunbergii after 48 h; however, it was not detected in non-mycorrhized P. thunbergii. The essential element Cu exhibited similar results as the non-essential element Cd. Furthermore, two species ECM fungi Suillus accelerates the uptake and transport of Cd in the host plant P. thunbergii.

Seagrass beds are important coastal ecosystems that provide valuable ecosystem services; however, they are increasingly threatened by climate change and human activities. We compared the fine-scale genetic structure of two tropical seagrass species, Enhalus acoroides and Cymodocea rotundata, which differ in their reproductive strategies, using 45 & times; 45 m plots with grid points at 5-m intervals. In addition, we compared the genetic structure between healthy and disturbed sites to better understand the mechanisms of population maintenance under different environmental conditions. Using high-resolution microsatellite markers, we conducted population genetic analyses of 732 samples of E. acoroides from nine plots and 621 samples of C. rotundata from eight plots in Bolinao (disturbed), as well as 665 samples of C. rotundata from eight plots in Laguindingan (undisturbed), Philippines. We detected higher clonal diversity and stronger genetic structure among plots in C. rotundata than in E. acoroides. Interestingly, multilocus genotypes of C. rotundata were shared between plots separated by up to approximately 3.58 km. We infer that C. rotundata populations have been maintained primarily through asexual reproduction, such as rhizome elongation and fragmentation. Furthermore, in C. rotundata, clonal diversity was higher and kinship was stronger at the disturbed site. Our findings indicate that C. rotundata is more vulnerable in highly disturbed areas and requires conservation and restoration efforts. Moreover, our results highlight the necessity of multiple sampling plots within a site to adequately estimate spatial genetic structure for conservation genetic studies of seagrasses.

Ectomycorrhizal (ECM) fungi preferentially form symbiotic associations with many woody plants. However, the genes regulating ECM formation and their functions in host plants remain poorly understood. In this study, PtoCAF1I gene from Populus tomentosa was isolated and functionally characterized. Bioinformatics analysis revealed that PtoCAF1I contains a conserved RNase D structural domain. Tissue expression analysis showed that PtoCAF1I was more highly expressed in roots than other tissues (leaves and stems). Overexpression of PtoCAF1I in P. tomentosa resulted in significantly greater shoot height, number of branches, and biomass in potted seedlings compared with wild-type (WT) plants. Inoculation experiments revealed that overexpression of PtoCAF1I significantly increased the total ECM rate compared with WT poplar inoculated with Cenococcum geophilum, whereas PtoCAF1I RNA interference (RNAi) lines presented an opposite trend. These results suggest that PtoCAF1I not only promotes poplar growth and branch development but also enhances the formation of ECM roots upon inoculation with C. geophilum.

Pine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (Rhabditida: Aphelenchoididae), results in significant global economic and ecological impacts. Although the Cry13Aa1 toxin from Bacillus thuringiensis shows nematicidal activity, its mechanism of action against B. xylophilus remains unclear. This study aimed to identify and characterize the receptors for Cry13Aa1 in B. xylophilus. We cloned the cDNAs encoding an Snf7 domain-containing protein (BxSnf7) from B. xylophilus. Far-western blot analysis revealed a specific binding interaction between BxSnf7 and Cry13Aa1, showing a dissociation constant (Kd) of 20.8 ± 4.2 nM. Interestingly, bioassay results indicated that silencing BxSnf7 increased the susceptibility of nematodes to Cry13Aa1 at higher concentrations, although the difference was not statistically significant. Besides, the combined application of BxSnf7 with Cry13Aa1 significantly enhanced nematicidal mortality (95.9 %) after 24 h of treatment, which higher than the expected mortality (42.8 %) (χ2 = 16.118, P = 0.048), indicating that the exogenous BxSnf7 synergistically enhances the activity of Cry13Aa1 toxin. These findings identify BxSnf7 as a novel Cry13Aa1 binding protein and reveal a unique mechanism by which BxSnf7 synergistically enhances the activity of Cry13Aa1. However, BxSnf7 does not function as the primary receptor, and further research is needed to investigate its role in modulating nematode susceptibility to Cry13Aa1.

Ectomycorrhizal (ECM) symbiosis can alter root tip structures; but the detailed mechanisms behind these changes and their impact on overall root architecture remain unclear. This study aimed to investigate the effects of ECM inoculation with Cenococcum geophilum (Cg) and Pisolithus orientalis (Po) on root growth, nutrient levels in shoots, and enzyme activities in soil for Chinese chestnut (Castanea mollissima) and pecan (Carya illinoinensis) seedlings. Chestnut and pecan seedlings were inoculated with Cg or Po. The growth parameters, including root and shoot development, were assessed. Nutrient levels of nitrogen, phosphorus, and potassium in the shoots were measured, and soil enzyme activities related to carbon, nitrogen, and phosphorus cycles, such as β-glucosidase, peroxidase, N-acetylglucosaminidase, and acid phosphatase, were quantified. Inoculation with ECM fungi significantly enhanced root growth, especially fine roots, and improved nutrient accumulation in the shoots of both seedlings. Cg and Po inoculation increased nitrogen, phosphorus, and potassium levels in the shoots. Moreover, the soil enzymes involved in the carbon, nitrogen, and phosphorus cycles, such as β-glucosidase, peroxidase, N-acetylglucosaminidase, and acid phosphatase, showed elevated activity levels under ECM inoculation. Notably, Cg inoculation elevated peroxidase activity, which is linked to root development and soil hormone regulation. ECM symbiosis, especially with Cenococcum geophilum, positively influences root structure and nutrient absorption, enhancing seedling growth in chestnut and pecan. These findings highlight the role of ECM fungi in improving plant growth, particularly under conditions of limited colonization.

The App6Aa2 toxin, derived from Bacillus thuringiensis, is nematicidal and highly toxic to Bursaphelenchus xylophilus. Receptors play a critical role in the mechanism of B. thuringiensis crystal toxin toxicity, yet the specific binding receptors for App6Aa2 in B. xylophilus have not been identified. This study identified a GPI-anchored protein, alpha-galactosidase (BxGal), in B. xylophilus as a potential binding protein. Western blotting and ELISA assays confirmed a high binding affinity between BxGal and App6Aa2 (Kd = 24.5 +/- 11.2 nM). Remarkably, combining App6Aa2 with the BxGal protein produced a synergistic effect, significantly increasing nematode mortality from 25 % to 81 % (P < 0.05), thereby enhancing the toxicity of App6Aa2 against B. xylophilus. Besides, RNAi silencing of BxGal in nematodes had no specific effect on App6Aa2 toxicity at high concentrations, while mortality increased slightly at lower concentrations. These findings indicate that BxGal is a high-affinity binding protein for App6Aa2; it does not function as the primary receptor and warrants further investigation into its role in modulating nematode susceptibility to App6Aa2.

Cenococcum geophilum is among the most globally abundant ectomycorrhizal (ECM) fungi and often dominates the root systems of woody plants, particularly in extreme environments. Therefore, C. geophilum is an ideal model for studying genetic diversity and adaptation of ectomycorrhizal symbiosis. In this study, we constructed a C. geophilum pan-genome using six high-quality whole-genome sequences representing the global diversity and found that dispensable genes are important for its environmental adaptation. We identified millions of single-nucleotide polymorphisms, hundreds of thousands of small insertions/deletions, 0.04–0.2% structural variation, and 21 horizontally transferred genes from non-host plants. Based on resequencing data from 304 global isolates, we investigated the geographic structure of C. geophilum populations. Genome-wide association and transcriptome studies identified 161 genomic regions significantly associated with nine biological and environmental adaption characteristics, including 2738 candidate genes such as EVM0002574 for cadmium, salt, and high temperature stresses. These reference genomes and diversity datasets provide resources and a comparative genomic platform for the study of ectomycorrhizal symbioses.

Ectomycorrhiza (ECM) significantly influences the establishment and stability of forest ecosystems. While evaluating ECM colonization is important for ECM symbiosis research, conventional methods using microscopes prove both time-intensive and subjective. To overcome this, we developed a high throughput and objective method for evaluating ECM colonization in a target tree under laboratory conditions by using quantitative PCR (qPCR). Primers specific for Populus tomentosa, Cenococcum geophilum (Cg), and Laccaria japonica (Lj) were designed and validated. After separately inoculating Cg and Lj, as well as co-inoculating Cg and Lj to P. tomentosa, the root samples were harvested at three different time points; 10, 20, and 40 days post-inoculation. ECM colonization was assessed by conventional microscopic method and qPCR with the designed primers. The designed primers showed a high specificity and quantitativity for each species, indicating their practical use in quantifying individual ECM species in tripartite conditions. Both ectomycorrhizal species showed a positive correlation between the conventional method and the qPCR method: Cg (Spearman correlation = 0.917, p < 0.001), Lj (Spearman correlation = 0.690, p < 0.01). Therefore, by using highly species-specific primers, we established a method to evaluate ECM colonization in vitro by qPCR, which can be an alternative and solution to the conventional method.

The impact of drought stress on plant growth in arid regions is a critical concern, necessitating the exploration of strategies to enhance plant drought resistance, particularly during the early stages of drought stress. This study focuses on the ectomycorrhizal fungus Cenococcum geophilum, renowned for its extensive genetic diversity and broad host compatibility, making it a crucial ally for host plants facing external stresses. We utilized Pinus massoniana seedlings inoculated with different ecotypic strains of C. geophilum under drought stress. The results showed that the inoculation of most strains of C. geophilum enhanced the drought resistance of P. massoniana seedlings under the early stages of drought stress, by influencing the water content, photosynthesis, accumulation of osmotic adjustment substances, and antioxidant enzyme activities in both shoots and roots of seedlings. Transcriptome analysis showed that mycorrhizal seedlings mainly regulated energy metabolism and reduction–oxidation reaction to resist early drought stress. Notably, the level of drought resistance observed in mycorrhizal seedlings was irrespective of the level of drought tolerance of C. geophilum strains. This study contributes essential data for understanding the drought response mechanisms of mycorrhizal P. massoniana seedlings inoculated by distinct C. geophilum ecotypes and guidance on selecting candidate species of ectomycorrhizal fungi for mycorrhizal afforestation in drought areas.

Seagrass beds are ecologically and economically important coastal ecosystems, and seagrass-associated organisms are a key part of their biodiversity. Marine organisms that reproduce through broadcast spawning are likely to have less genetic differentiation among populations than those that use other modes of reproduction, but this has not been well studied. Here, we investigated the genetic diversity, genetic differentiation, and migration patterns of the seagrass-associated sea star Protoreaster nodosus across 12 sites spanning approximately 2500 km from the Ryukyu Archipelago, Japan, to the Philippines. We genotyped 405 individuals by using seven microsatellite loci and analyzed allelic richness and expected heterozygosity as indices of genetic diversity. Of these two indices, only expected heterozygosity decreased slightly with increasing latitude. These results suggest that genetic diversity has not clearly decreased, even in the isolated Ryukyu Archipelago populations. Geographic distance was significantly correlated with genetic differentiation (pairwise FST: − 0.005 to 0.049). However, populations in the Ryukyu Archipelago and the Philippines showed relatively low genetic structuring and the pairwise genetic differentiation between these regions was often non-significant. Analysis of historical migration rates showed bidirectional north–south migration, which appears to be influenced by the Kuroshio Current and its countercurrents.

Zinc (Zn) deficiency is a significant nutritional limitation to crop yield globally, particularly in calcareous soil environments. Tree peony of Peaonia ostii ‘Fengdan’ is regarded as an oil crop due to its seeds rich in alpha-linolenic acid, a beneficial compound for health promotion. However, low seed yield remains a primary challenge in attaining sufficient seed oil from tree peony. In this study, Zn fertilization was applied to soil or foliage of P. ostii ‘Fengdan’ in the growth period before fruit development. Our findings reveal that foliar Zn-spraying, as opposed to soil application, proves to be a more effective method for augmenting seed yield, Zn accumulation and photosynthetic capacity in ‘Fengdan’. Comparative analyses of the leaf proteome of ‘Fengdan’ using iTRAQ profiling under foliar Zn-spraying identified 115 differentially expressed proteins (DEPs), including 36 upregulated proteins, which likely contribute to the observed increase in seed yields of ‘Fengdan’ caused by foliage Zn-spraying. Specifically, Zn2+ stimulation of phosphatidylinositol signaling initiates a cascade of metabolic regulations. Firstly, ATP synthesis promotes leaf photosynthetic capacity, facilitated by improved sucrose metabolism through upregulated pullulanase and 1,4-alpha-glucan-branching enzyme. Furthermore, lipid synthesis and transport are facilitated by upregulated lipoyl synthase and plastid lipid-associated proteins. Additionally, DEPs involved in secondary metabolism are upregulated in the production of various metabolites conducive to ‘Fengdan’ growth. Overall, our results demonstrate that foliage Zn-spraying enhances seed yield in P. ostii ‘Fengdan’ by elevating Zn content and secondary metabolite synthesis in leaves, thereby augmenting leaf photosynthetic capacity and lipid synthesis. This study provides an effective way to increase seed yield of tree peony by exogenous Zn application.

The application of mycorrhizal fungi as a bioaugmentation technology for phytoremediation of heavy metal (HM) contaminated soil has attracted widespread attention. In order to explore whether the adaptation of Pinus massoniana (P. massoniana) to metal polluted soil depends on the metal adaptation potential of their associated ectomycorrhizal fungi (ECMF), we evaluated the cadmium (Cd) tolerance of 10 ecotypes of Cenococcum geophilum (C. geophilum) through a membership function method, and P. massoniana seedlings were not (NM) or inoculated by Cd non-tolerant type (JaCg144), low-tolerant (JaCg32, JaCg151) and high-tolerant (JaCg205) isolates of C. geophilum were exposed to 0 and 100 mg·kg-1 for 3 months. The result showed that, each ecotype of C. geophilum significantly promoted the growth, photosynthesis and chlorophyll content, proline (Pro) content and the activity of peroxidase (POD) of P. massoniana seedlings, and decreased malonaldehyde (MDA) content and catalase (CAT) and superoxide dismutase (SOD) activity. The comprehensive evaluation D value of the tolerance to Cd stress showed that the order of the displaced Cd resistance of the four ecotypic mycorrhizal P. massoniana was: JaCg144 > JaCg151 > JaCg32 > JaCg205. Pearson correlation analysis showed that the Sig. value of the comprehensive evaluation (D) values of the strains and mycorrhizal seedlings was 0.077 > 0.05, indicating that the Cd tolerance of the the C. geophilum isolates did not affect its regulatory effect on the Cd tolerance of the host plant. JaCg144 and JaCg151 which are non-tolerant and low-tolerant ecotype significantly increased the Cd content in the shoots and roots by about 136.64-181.75% and 153.75-162.35%, indicating that JaCg144 and JaCg151 were able to effectively increase the enrichment of Cd from the soil to the root. Transcriptome results confirmed that C. geophilum increased the P. massoniana tolerance to Cd stress through promoting antioxidant enzyme activity, photosynthesis, and lipid and carbohydrate synthesis metabolism. The present study suggests that mental-non-tolerant ecotypes of ECMF can protect plants from Cd pollution, providing more feasible strategies for ectomycorrhizal-assisted phytoremediation.

The studied fifteen populations of Cymodocea rotundata and Enhalus acoroides in Mindanao, southern Philippines revealed a remarkable strategy for reproduction and dispersal. E. acoroides populations exhibits high rates of sexual recruitment with high clonal richness ranged from 0.9 to 1.0, while C. rotundata exhibited monoclonal to high clonal richness ranged from 0 to 1.0. Overall, clonal richness was high indicating sexual reproduction is prevalent, but varied for C. rotundata. The largest genet found in the study was at Rizal (RIZ), northern Mindanao wherein throughout the sampling area only one genet was identified, maybe influenced by environmental condition and hydrodynamic of the water currents and tidal fluctuation in this range-edge population. Significant pairwise population genetic differentiation was found among many sites. Isolation by distance (IBD) was detected in C. rotundata (P < 0.05) but not in E. acoroides (P = 0.253). IBD manifested among populations maybe influenced by the ocean currents in Mindanao, but it has to be confirmed yet in the future study. The results have implications for understanding on how the environmental conditions can influenced the dispersal strategy of these species in natural settings. These species manifested high plasticity most likely to adopt to environmental conditions favoring natural selection for long term demographic stability and fitness. The genetic diversity, structure, reproduction and dispersal strategies observed in this study may prove useful for eventual genetic conservation plans of these two foundational seagrass species.

Many marine species have a pelagic larval phase that undergo dispersal among habitats. Studies on marine larval dispersal have revealed a large variation in the spatial scale of dispersal, and accumulated evidence has shown that seascape patchiness is the major determinant for variation in self-recruitment. However, few studies have investigated the influence of geographic settings on marine larval dispersal. Bays or lagoons generally enhance the retention of larvae, while larvae are more likely to be flushed by strong currents in open coasts. To examine associations between larval dispersal, geographic setting, and hydrodynamics, we compared fin-scale dispersal patterns, self-recruitment, and local retention of two anemonefishes ( Amphiprion frenatus and A. perideraion ) between a semi-enclosed bay and an open coast in the Philippines combining genetic parentage analysis and biophysical dispersal modelling. Contrary to our expectations, parentage analysis revealed lower estimates of self-recruitment in the semi-closed bay (0–2%) than in the open coast (14–15%). The result was consistent with dispersal simulations predicting lower local retention and self-recruitment in the former (0.4% and 19%) than in the latter (2.9% and 38%). Dispersal modelling also showed that cross-shore currents toward offshore were much stronger around the semi-closed bay and were negatively correlated with local retention and self-recruitment. These results suggest that stronger cross-shore currents around the semi-closed bay transport anemonefish larvae to the offshore and mainly contributed to the lower self-recruitment. Our results highlight difficulty in predicting self-recruitment from geographic setting alone and importance of hydrodynamics on it.

AbstractCenococcum geophilum (C. geophilum) is a widely distributed ectomycorrhizal fungus that plays a crucial role in forest ecosystems worldwide. However, the specific ecological factors influencing its global distribution and how climate change will affect its range are still relatively unknown. In this study, we used the MaxEnt model optimized with the kuenm package to simulate changes in the distribution pattern of C. geophilum from the Last Glacial Maximum to the future based on 164 global distribution records and 17 environmental variables and investigated the key environmental factors influencing its distribution. We employed the optimal parameter combination of RM = 4 and FC = QPH, resulting in a highly accurate predictive model. Our study clearly shows that the mean temperature of the coldest quarter and annual precipitation are the key environmental factors influencing the suitable habitats of C. geophilum. Currently, appropriate habitats of C. geophilum are mainly distributed in eastern Asia, west‐central Europe, the western seaboard and eastern regions of North America, and southeastern Australia, covering a total area of approximately 36,578,300 km2 globally. During the Last Glacial Maximum and the mid‐Holocene, C. geophilum had a much smaller distribution area, being mainly concentrated in the Qinling‐Huaihe Line region of China and eastern Peninsular Malaysia. As global warming continues, the future suitable habitat for C. geophilum is projected to shift northward, leading to an expected expansion of the suitable area from 9.21% to 21.02%. This study provides a theoretical foundation for global conservation efforts and biogeographic understanding of C. geophilum, offering new insights into its distribution patterns and evolutionary trends.

Seagrasses are marine angiosperms, and seagrass beds maintain the species diversity of tropical and subtropical coastal ecosystems. For proper understanding, management and conservation of coastal ecosystems, it is essential to understand seagrass population dynamics. Population genetic studies can cover large geographic scales and contribute to a comprehensive understanding of reproductive dynamics and potential dispersal among locations. The clonal and genetic diversity and genetic connectivity of Thalassia hemprichii in the Philippines were estimated by a population genetics approach. The geographic scale of this study has a direct distance of approximately 1600 km. Although high clonal diversity was found in some sites (R = 0.07–1.00), both sexual and asexual reproduction generally maintains separate populations. Genetic diversity is not definitely correlated with latitude, and genetic differentiation is significant in all pairs of sites (FST = 0.026–0.744). Complex genetic structure was found in some regions, even at a fine geographic scale. The migration of fruits and seedlings was elucidated as an infrequent and stochastic event. These results suggest the necessity for the conservation of this species due to a deficiency in migrants from external regions.

Pine wilt disease (PWD), Bursaphelenchus xylophilus, is an extremely threatening invasion forest disease throughout the world, especially in Asia. B. xylophilus is spread in Asia by vector beetles of Monochamus alternatus, which has long no effective control method. Understanding of landscape effects on the dispersal and outbreaks of forest pests is crucial to establishing effective ecological control strategies. Here, we analyzed the samples of M. alternatus collected in a fine-scale in order to estimate the effects of landscape types on the genetic structure and dispersal of M. alternatus. The landscapes included the geographical scales, forest types and land uses. The individuals of M. alternatus were genotyped by using whole-genome resequencing. Population genetic structures were clearly differentiated at the range of 250 kms, suggesting the intermediate scale is an effective barrier against natural dispersal of M. alternatus. We used the least-cost distances, least-cost transect analysis, and distance-based redundancy analysis to estimate the effects of forest types and land uses within the fine scales. The results showed that the gene flow and genetic diversity were positively correlated with host and mixed forests, whereas negatively with non-host forests. Among land-use landscapes, the roads had the positive effect on gene flow and genetic diversity but farmland and urban uses had negative effects. This highlights that human-mediated transport via roads was likely to be the main factor leading to the long-distance invasion of M. alternatus, whereas non-host landscapes could suppress the spread of this species. These findings may be useful to control the PWD dispersed by M. alternatus.