The endoplasmic reticulum (ER) is a central hub coordinating protein homeostasis and lipid metabolism in eukaryotic cells. In microalgae, which inhabit highly fluctuating environments, ER stress is increasingly recognize...The endoplasmic reticulum (ER) is a central hub coordinating protein homeostasis and lipid metabolism in eukaryotic cells. In microalgae, which inhabit highly fluctuating environments, ER stress is increasingly recognized as a driver of lipid remodeling rather than a secondary metabolic consequence. This review synthesizes recent advances in ER stress signaling in microalgae, focusing on Chlamydomonas reinhardtii, and places these findings in a comparative eukaryotic context. Microalgae retain a conserved, IRE1-centered unfolded protein response (UPR) while lacking auxiliary branches found in animals and land plants. Activation of ER stress induces extensive reprogramming of membrane lipid composition, fatty acid desaturation, sterol metabolism, and triacylglycerol (TAG) accumulation. Notably, the Chlamydomonas IRE1/bZIP1 pathway functions to restrain excessive TAG accumulation, thereby prioritizing membrane adaptation and ER homeostasis. The graded and dynamic nature of this response likely compensates for the simplified single-sensor architecture by enabling flexible modulation of downstream outputs depending on stress intensity and duration. Importantly, ER stress responses exhibit distinct modes depending on stress severity: moderate stress promotes adaptive membrane stabilization, whereas severe or prolonged stress redirects membrane-derived fatty acids into TAG for sequestration. This lipid-centered adaptation contrasts with land plants, which stabilize membrane composition without substantial TAG accumulation, and with yeast and animals, where membrane biogenesis and neutral lipid storage occur in parallel, with distinct regulatory features. By integrating insights across eukaryotes, this review highlights ER stress as a framework for understanding lipid remodeling in microalgae and discusses how UPR manipulation may enable rational engineering of lipid production platforms.
Plants perceive danger signals, pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), to activate immune responses such as transient apoplastic alkalinization and reactive oxygen species (ROS) production. Howe...Plants perceive danger signals, pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), to activate immune responses such as transient apoplastic alkalinization and reactive oxygen species (ROS) production. However, how these pH and redox changes occur across organs and tissues during pattern-triggered immunity (PTI) remains poorly understood. Using genetically-encoded biosensors (GEBs), we monitored cytosolic pH and redox dynamics across whole Arabidopsis seedlings with spatiotemporal resolution. Global treatments with diverse danger signals first induced cytosolic acidification and oxidation in roots, followed by petioles and, later, the hypocotyl, revealing organ-specific responsiveness and a bidirectional response gradient. By contrast, Pseudomonas syringae pv. tomato (Pto) DC3000 induced sustained cytosolic alkalinization and suppressed redox responses in inoculated shoots, even when co-treated with PAMPs. Mutants either lacking a functional flagellum (ΔfliC) or Type-3 secretion system (ΔhrcC) induced opposite or no long-term responses in the cytosol, respectively. Together, these findings highlight that the plant's immune response is not uniform but instead follow organ- and tissue-specific patterns which are trigger-dependent, and reflect distinct capacities of seedling cells to activate PTI.
Disentangling genotype × environment (G×E) controls of flowering time requires phenotypes that link molecular regulation, developmental physiology and environment. Here, we integrated time-resolved measurements of apical...Disentangling genotype × environment (G×E) controls of flowering time requires phenotypes that link molecular regulation, developmental physiology and environment. Here, we integrated time-resolved measurements of apical development, final leaf number (FLN), and expression of the flowering-time genes VRN1, VRN2 and VRN3 across contrasting temperature and photoperiod regimes in six wheat genotypes spanning a wide range of developmental sensitivities. By combining controlled-environment phenotyping with concurrent gene-expression profiling, we show that environmentally driven variation in FLN is coherently explained by shifts in the timing of key apical transitions and associated VRN gene-expression dynamics. These integrated datasets were used to parameterise and interrogate the Cereal Anthesis Molecular Phenology (CAMP) model, enabling direct comparison between observed foliar gene-expression time courses and modelled gene activity. While overall developmental responses were well captured by the model, systematic differences between observed and modelled gene-expression patterns highlight the importance of distinguishing foliar expression from apical regulatory activity, as well as differences in temporal scaling. Building on this framework, we present a phenotyping protocol based on FLN responses to defined temperature and photoperiod treatments that delivers unconfounded developmental phenotypes explicitly linked to underlying genetic regulation.
Multiplex editing is crucial for analyzing complex multiple-gene traits in woody plants, yet its application remains limited by low transformation efficiency and lengthy regeneration cycles. To overcome these barriers, t...Multiplex editing is crucial for analyzing complex multiple-gene traits in woody plants, yet its application remains limited by low transformation efficiency and lengthy regeneration cycles. To overcome these barriers, this study establishes an efficient protoplast isolation protocol for Pyrus, employing 1.0% cellulase R10 and 0.4% macerozyme R10 with an 8.5 h digestion, and demonstrates its broad applicability across seven economically important woody plants. Coupling a 40% PEG-4000-mediated transfection regimen with DNA-free CRISPR/Cas9 ribonucleoprotein (RNP) delivery enabled multiplex genome editing in isolated protoplasts. Using this platform, simultaneous disruption of PbrARC3, PbrPARC6, and PbrFtsZ2-1a, key components of the chloroplast division apparatus, consistently reproduced macro-chloroplast abnormalities, confirming effective multigene perturbation within a single cellular context. Notably, chloroplast division failure activated chloroplast-to-nucleus retrograde signaling, evidenced by induction of nuclear stress-response genes PbrRBOHD and PbrZAT12, a concomitant surge in reactive oxygen species, and progression to severe cellular deformation. These results establish a rapid, cross-genus protoplast-RNP workflow that enables DNA-free multiplex editing and accelerates genotype-to-phenotype analyses in woody perennials. The approach provides a practical foundation for functional genomics and supports advances in non-transgenic precision breeding of tree crops.
Lipid droplets (LDs) are ubiquitous eukaryotic organelles for neutral lipid storage and have long been studied primarily in seeds for storing carbon and energy. However, accumulating evidence indicates that LDs are also...Lipid droplets (LDs) are ubiquitous eukaryotic organelles for neutral lipid storage and have long been studied primarily in seeds for storing carbon and energy. However, accumulating evidence indicates that LDs are also abundant and highly dynamic in vegetative tissues such as leaves. In Arabidopsis thaliana, diverse abiotic and biotic stresses induce triacylglycerol accumulation and LD formation in leaves. Recent advances in lipid metabolism research, mass spectrometry-based proteomics, and genetic analysis have substantially advanced our understanding of leaf LD biogenesis, composition, and function. Proteomic studies have identified numerous LD-localized proteins, including regulators of LD formation, enzymes involved in lipid synthesis and modification, cytoskeleton-associated proteins, and factors linked to stress responses and protein quality control. In parallel, analyses of mutants with altered LD accumulation have revealed key roles for lipid trafficking, sterol homeostasis, carbon allocation, and peroxisomal lipid degradation. Together, these findings redefine leaf LDs as multifunctional organelles that integrate lipid metabolism with environmental stress responses and cellular homeostasis. This review summarizes recent progress in identifying LD-associated proteins and genetic regulators to discuss emerging concepts regarding the diverse roles of LDs in plant leaves.
Plant oils are widely used in the food, fuel, and oleochemical industries, with their chemical properties and applications largely determined by fatty acid composition. Phosphatidylcholine:Diacylglycerol Cholinephosphotr...Plant oils are widely used in the food, fuel, and oleochemical industries, with their chemical properties and applications largely determined by fatty acid composition. Phosphatidylcholine:Diacylglycerol Cholinephosphotransferase (PDCT), a plant-exclusive, multi-spanning transmembrane enzyme, catalyzes the interconversion between diacylglycerol and phosphatidylcholine and plays a key role in shaping oil fatty acid profiles in plants. Despite its importance, the structure-function relationship of PDCT remains poorly understood. Our recent structural modelling suggests that PDCT may function as a dimer through swapping of the N-terminal region between protomers. In this study, we used soybean (Glycine max) PDCT1 to further examine the terminal regions and key residues in the predicted swapped N-terminal transmembrane region required for PDCT activity. Our results reveal that the disordered region at the N-terminus, and the less-conserved C-terminus, are both dispensable for catalysis, while conserved residues Glu75 and Asp89 within the swapped N-terminal transmembrane region are essential for enzyme activity and/or stability. Notably, co-expression of the inactive E75A N-terminal mutant and the inactive D230A C-terminal catalytic site mutant partially restored PDCT activity, providing support for the proposed domain-swapping mechanism. Together, this study provides new insights into the catalytic mechanisms of PDCT and may inform future efforts to exploit PDCT in plant oil engineering.
Stable rice production is continuously threatened by rapidly evolving, devastating rice pathogens, underscoring the need for new sources of disease resistance. Phosphoinositide 4-kinases (PI4Ks) are important in the phos...Stable rice production is continuously threatened by rapidly evolving, devastating rice pathogens, underscoring the need for new sources of disease resistance. Phosphoinositide 4-kinases (PI4Ks) are important in the phosphoinositide biosynthetic pathway and are classified into Type II and Type III. Type III PI4Ks have been studied for their role in plant immunity; however, the function of Type II PI4Ks remains underexplored. In this study, we investigated the role of type II PI4Ks, OsPI4Kγ2 and OsPI4Kγ6 in rice immunity. Using genome editing, we generated knockout mutants of OsPI4Kγ2 and OsPI4Kγ6, which displayed enhanced resistance to rice blast and bacterial blight as indicated by reduced disease symptoms, increased production of reactive oxygen species (ROS), and elevated defense-related gene expression. Moreover, loss of OsPI4Kγ2 or OsPI4Kγ6 significantly reduced PtdIns4P levels and biotrophic interfacial complex (BIC) formation rates, indicating that these kinases are required for maintaining normal PtdIns4P homeostasis and infection-related structure formation in rice. This study thus provides evidence that OsPI4Kγ2 and OsPI4Kγ6 act as negative regulators of rice immunity, offering new insights into their role in plant defense.
WRKY Transcription factors (TFs) are central regulators of plant growth, development, and responses to biotic and abiotic stresses. In rice, the large size of this family, coupled with structural diversity, multifunction...WRKY Transcription factors (TFs) are central regulators of plant growth, development, and responses to biotic and abiotic stresses. In rice, the large size of this family, coupled with structural diversity, multifunctionality, redundancy, and extensive network integration, complicates efforts to define precise gene functions. While these features confer regulatory robustness, they limit the interpretability of single-gene analyses and obscure direct gene-trait relationships. This review examines the challenges underlying WRKY functional characterization in rice and highlights how they arise from the intrinsic properties of this gene family. We then present a challenge-centered framework in which experimental and computational approaches are mapped onto the four major barriers to WRKY functional resolution, such as context-dependent activity, functional redundancy, and target specificity. By emphasizing how these approaches resolve distinct layers of WRKY complexity, we provide a problem-driven synthesis of current methodologies. We further underscore the need for integrated WRKY-centric databases to support hypothesis generation and accelerate functional discovery. A systems-level understanding of WRKY TFs, grounded in their regulatory complexity, will be essential to harness their potential to improve stress resilience and productivity in rice.
Improving plant growth and stress tolerance is a significant challenge that affects crop production. Here, we demonstrate that ethylene pretreatment in a critical period during germination accelerates development and lea...Improving plant growth and stress tolerance is a significant challenge that affects crop production. Here, we demonstrate that ethylene pretreatment in a critical period during germination accelerates development and leads to larger cotyledons, leaves, and roots with larger cells. Additionally, ethylene pretreatment led to prolonged increases in the number of chloroplasts per cell, larger chloroplasts, and higher CO2 assimilation. All of these responses were eliminated in mutants lacking two cytosolic invertases (CINV1, CINV2). Time-lapse imaging showed that wild-type plants have an initial transient increase in root growth within several hours of ethylene removal and transfer to light. This was followed by a second prolonged increase in growth. The cinv1;cinv2 mutants retained an initial, rapid increase in root growth, but lacked prolonged growth stimulation, indicating that there are two phases to the growth stimulation after ethylene pretreatment, with the second, prolonged phase requiring these invertases. RNA-sequencing and untargeted metabolomics revealed that the cinv1;cinv2 mutants still had responses to ethylene pretreatment, but these responses were attenuated and diverged from wild-type. Thus, these invertases are required for changes in chloroplast number and size, increased photosynthesis, and prolonged stimulation of growth after ethylene pretreatment. Ethylene pretreatment represents a possible new approach to explore as a way to increase plant growth.
The apoplast of leaves is involved in nutrient transport, microbe-host interactions, systemic signaling, cell wall dynamics, and serves as an interface for various other physiological processes. The composition of the ap...The apoplast of leaves is involved in nutrient transport, microbe-host interactions, systemic signaling, cell wall dynamics, and serves as an interface for various other physiological processes. The composition of the apoplastic solute pool, which supports many of these functions, is dynamic and shaped by developmental and environmental cues. However, due to the complexity and compartmentalization of the apoplast, analysing these fluids - and thus the associated physiological processes - remains technically challenging. This study introduces a minimally invasive method for extracting apoplastic fluids from leaves of selected dicots (e.g. Arabidopsis thaliana, Vicia faba, and many more), offering two key advantages: (i) repeated extractions from the same leaves to generate time-series data, such as every 24 hours, over consecutive days, and (ii) high spatial resolution, enabling identification of macrodomains within the leaf apoplast. For example, abscisic acid macrodomains were revealed along the leaf axis, providing insight into apoplastic hormone regulation. The method also reveals other previously unrecognized aspects, such as the accumulation of kaempferol glycosides in the apoplast after plants experienced salt stress. Finally, the method addresses the distortion of apoplast compound levels caused by dilution bias that results from the inconsistent recovery of infiltration fluid. Adding pyranine enables correction, ensuring accurate and comparable data. By integrating spatial and temporal precision, this new tool will promote a deeper understanding of plant apoplastic processes and their physiological relevance in various biological contexts.
The plant hormone ethylene is perceived by a small family of histidine kinase-like ethylene receptor proteins in Arabidopsis. The five receptors, considered functionally redundant, are structurally categorized into two s...The plant hormone ethylene is perceived by a small family of histidine kinase-like ethylene receptor proteins in Arabidopsis. The five receptors, considered functionally redundant, are structurally categorized into two subfamilies. Little is known about the biological importance of subfamily classification in receptor signaling or the degree of redundancy. By testing the genetic interactions of receptor genes, our results annotated differential emergent signaling of receptor isoforms. Except for the ethylene-insensitive ETR1-1 receptor, ethylene-insensitive receptors require other wild-type isoforms to convey receptor signaling. The two subfamilies are mutually required for efficient receptor signaling, and emergent receptor signaling is minimal within a subfamily. The receptor signal output was minimal for the subfamily I member ETR1, barely detectable for ERS1, and relatively strong for ETR1 and differential for ERS1 in combination with a subfamily II member. ETR1 has unique roles in receptor signaling. Together with other lines of evidence, our findings imply a low degree of functional interchangeability among receptor isoforms. The low degree of functional redundancy thus enriches the heterogeneity of receptor complexes, enabling an extended range of ethylene responsiveness. The structural features of plant ethylene receptor-like proteins were analyzed, shedding light on the evolution and emergent properties of ethylene receptor-like proteins.
Kin recognition enables plants to optimize growth strategies when competing with heterospecific neighbors, yet its prevalence in herbicide-resistant weeds and how herbicide-resistant weeds exploit kinship to interact wit...Kin recognition enables plants to optimize growth strategies when competing with heterospecific neighbors, yet its prevalence in herbicide-resistant weeds and how herbicide-resistant weeds exploit kinship to interact with allelopathic crops remain largely unknown. Here, we experimentally demonstrate that kin recognition occurred in 4 out of 10 tested herbicide-resistant weed biotypes from wheat and paddy fields. Root segregation treatments confirmed that kin recognition was mediated by root-secreted chemical signals. When interacting with allelopathic rice and wheat, kin-recognition-enabled weed biotypes exhibited striking plasticity. Kinship-based pairing reduced root allocation but accelerated flowering and increased seed production. Crucially, herbicide-resistant weed biotypes growing with kin lowered exudation of the signaling (-)-loliolide, which reduced allelochemical production in crops and minimized crop-mediated growth inhibition. This cascade allowed weeds to reallocate resources toward reproduction and to reduce defense investment in crops-revealing a novel "signal-mediated mutual de-escalation" strategy. The findings suggest that kin recognition is an adaptive trait in herbicide-resistant weeds, reducing allelopathy from crops through (-)-loliolide-mediated chemical interactions. By modulating crop signaling systems, herbicide-resistant weeds exploit kinship for population expansion in agroecosystems, highlighting the role of kinship strategy in shaping weed adaptation to develop integrated weed management approaches.
In the context of advocacy for yield stability, trade-offs between yield and yield stability, the frequent lack of definitions, and the variation in methods when they are explicit, this essay connects two perspectives: p...In the context of advocacy for yield stability, trade-offs between yield and yield stability, the frequent lack of definitions, and the variation in methods when they are explicit, this essay connects two perspectives: phenotypic plasticity and factor analytics. Phenotypic plasticity brings over a century of research in developmental biology, ecology and evolution, and is gaining traction in crop science. Factor analytics is an advanced linear mixed model of multi-environment data with factor analytic variance structures for the genotype-by-environment interaction effect. We (1) review phenotypic plasticity, define agronomically adaptive plasticity where varieties (or practices) consistently return superior yield (or other traits) across environments with no trade-off, and describe percentile-plasticity plots to assess the agronomic value of plasticity; (2) outline factor analytic models, and (3) link phenotypic plasticity and factor analytic models mathematically and empirically. We show that phenotypic plasticity of cereal yield correlates positively with overall performance obtained from factor analytic models when plasticity is adaptive and negatively when it is maladaptive. We conclude that phenotypic plasticity contributes biological meaning to opaque analytical approaches, and that biologically grounded statistics are needed to challenge weak agronomic narratives, such as advocacy for stability that might reflect decision biases rather than critical consideration of its benefits.
Marine heatwaves (MHWs) are intensifying under climate change, yet the physiological limits that constrain seagrass resilience remain poorly defined. We experimentally tested the responses of the surfgrass Phyllospadix s...Marine heatwaves (MHWs) are intensifying under climate change, yet the physiological limits that constrain seagrass resilience remain poorly defined. We experimentally tested the responses of the surfgrass Phyllospadix scouleri, a foundation species of the Northeast Pacific coast, to simulated MHWs of contrasting intensity. In a 27-day mesocosm experiment, plants were exposed to fluctuating temperatures representing a severe MHW (23.5 ± 1.5 °C) and an extreme MHW (26.5 ± 1.5 °C), while photosynthetic performance, respiration, nitrogen metabolism, oxidative stress, and growth were monitored during and after warming. Phyllospadix scouleri maintained photosynthetic capacity and carbon balance under severe warming but exhibited pronounced physiological disruption at extreme temperatures, including sustained photoinhibition, reduced nitrate assimilation, elevated respiration, and negative daily productivity. These effects persisted after heat stress, leading to reduced growth and indicating incomplete recovery. Multivariate analyses revealed a transition from tolerance to functional breakdown near 26.5 °C, suggesting a potential physiological tipping point only 5-6 °C above current summer maxima in the area of the studied population. Our findings indicate that intensifying MHWs may rapidly erode the thermal safety margin of temperate seagrasses, pushing foundational coastal ecosystems toward metabolic instability under continued ocean warming.
As global food demand rises against the backdrop of environmental and health concerns from intensive agrochemical use, there is an urgent need for sustainable crop-management strategies. In this perspective, seed-borne e...As global food demand rises against the backdrop of environmental and health concerns from intensive agrochemical use, there is an urgent need for sustainable crop-management strategies. In this perspective, seed-borne endophytic microbes, including bacteria and fungi, in legumes offer a naturally inherited bioinoculant system. This review integrates 35 Scopus and Web of Science studies to examine the occurrence, transmission dynamics, and functional diversity of endophytes within seeds of key legume species. Seed endophytes contribute to plant development and productivity through multiple mechanisms: atmospheric nitrogen fixation; solubilization of phosphorus and potassium; synthesis of siderophores and indole-3-acetic acid; and modulation of rhizosphere microbial communities, collectively enhancing germination rate, biomass accumulation, and yield. Under abiotic stress conditions, such as drought, nutrient deficiency, or contaminant exposure (metals and pesticides), such beneficial microbes promote root architecture remodeling, exopolysaccharide secretion, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, thereby improving stress resilience. In biotic contexts, certain bacterial endophytes may contribute to biocontrol through antibiotic production, while fungal inoculants have been reported to synthesize alkaloids that can deter herbivores. By elucidating the multifaceted roles of legume seed endophytes, this review underscores their potential as a turnkey, eco-friendly bioinoculants, paving the way for greener agricultural practices without compromising crop performance.
Flooding is a major abiotic stress that restricts terrestrial plant growth and survival. A plant tissue's ability to avoid or sustain critical oxygen deprivation (hypoxia) and subsequent re-oxygenation damage is vital fo...Flooding is a major abiotic stress that restricts terrestrial plant growth and survival. A plant tissue's ability to avoid or sustain critical oxygen deprivation (hypoxia) and subsequent re-oxygenation damage is vital for its survival. Submergence triggers rapid ethylene and hypoxia signalling that in turn control acclimation responses, promoting plant resilience. Interestingly, an extensive range of additional environmental and internal factors were shown to influence these canonical signalling pathways associated with flooding acclimation and tolerance. Here, we discuss how such integrative ethylene- and hypoxia-dependent signalling enables plants to anticipate and prepare for potential flooding-induced hypoxia stress, fine-tune acclimation according to the environmental and internal metabolic context, and effectively orchestrate re-oxygenation responses. Furthermore, plants naturally experience environmental stress manifold throughout their lives, which may lead to long-term morphological adaptations and the encoding of stress memory to promote survival against sequential stressors.
Glucosinolates are sulfur-rich plant specialized metabolites with applications in nutraceutical and agricultural biotechnology. Microbial production of complex glucosinolates derived from chain-elongated amino acids rema...Glucosinolates are sulfur-rich plant specialized metabolites with applications in nutraceutical and agricultural biotechnology. Microbial production of complex glucosinolates derived from chain-elongated amino acids remains constrained by difficult-to-express key biosynthetic enzymes. Here, we report the first microbial biosynthesis of 2-phenylethyl glucosinolate (2PE) through a modular engineering strategy combining Escherichia coli and Saccharomyces cerevisiae. The bacterial module was critical to achieve functional expression of an iron-sulfur cluster enzyme that catalyzes amino acid side chain elongation of phenylalanine to homophenylalanine. The yeast module contained the core structure pathway converting homophenylalanine to 2PE. By optimizing the modules separately, we identified superior CYP79F, MAM, and BCAT variants among the tested brassicaceous enzymes. To address a metabolic bottleneck in the sulfation step catalyzed by a sulfotransferase, we optimized PAPS co-factor availability via sulfate feeding, resulting in a 10-fold increase in 2PE titers and partial alleviation of the desulfo-2-phenylethyl glucosinolate (ds-2PE) bottleneck. Further optimization of carbon source selection and autoinduction strategies enabled the first demonstration of 2PE biosynthesis from phenylalanine by combining the two modules. This work establishes a platform for microbial production of complex glucosinolates derived from chain-elongated amino acids via pathway modularization and co-culture engineering.
Woody plants such as grapevine (Vitis vinifera L.) are vulnerable to trunk diseases caused by wood pathogens that disrupt vascular function, and induce severe leaf symptoms associated with major metabolic disturbances an...Woody plants such as grapevine (Vitis vinifera L.) are vulnerable to trunk diseases caused by wood pathogens that disrupt vascular function, and induce severe leaf symptoms associated with major metabolic disturbances and canopy decline. Over time, these diseases can irreversibly alter the plant physiology and phenotype, ultimately reducing vine longevity. One of the most predominant trunk diseases is esca, which is a major cause of vineyard dieback, with rising incidence worldwide over the past decade. However, the molecular mechanisms underlying esca symptom development remain unclear. In this study, we leveraged the heterogeneous expression of esca-symptoms within individual grapevines to investigate molecular responses in both symptomatic and asymptomatic leaf tissues collected in the field. By combining metabolite profiling, RNA-seq and whole genome bisulfite sequencing, we show that metabolic alterations and extensive transcriptomic reprogramming are restricted to symptomatic leaves and are partially associated with local changes in DNA methylation. In addition, asymptomatic leaves display distinct DNA methylation changes, a few being shared with symptomatic tissues, suggesting a global plant epigenetic response to the disease. Notably, a subset of these methylation marks are observed prior to symptom development, highlighting the potential of epigenetic markers for the early detection of trunk diseases in perennial plants.