Exploratory profiling of microbial communities associated with tapping panel dryness in Hevea brasiliensis

Exploratory profiling of microbial communities associated with tapping panel dryness in Hevea brasiliensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploratory profiling of microbial communities associated with tapping panel dryness in Hevea brasiliensis Ann Tom, Sainamole Kurian P., Shaji Philip, Deepu Mathews, Reshmy Vijayaraghavan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8276740/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2026 Read the published version in Archives of Microbiology → Version 1 posted 7 You are reading this latest preprint version Abstract Tapping Panel Dryness is a complex physiological disorder in Hevea brasiliensis that leads to the cessation of latex flow, causing significant economic loss, yet its underlying cause remains unclear. Anatomical investigation of bark samples collected from TPD-affected samples exhibited deformed latex vessels, blocked sieve tubes, and DNA-containing bodies within phloem elements. Metagenomic profiling indicated largely similar microbial composition and diversity between healthy and TPD-affected bark samples, except for the presence of low-abundance taxa such as phytoplasma only in affected samples. However, predicted metabolic pathways differed significantly between healthy and TPD samples. The combined anatomical, cytological, and molecular evidences in the current study supports the potential involvement of phytoplasma in the etiology of TPD. Core microbiome Para rubber Phytoplasma Natural rubber TPD Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Hevea brasiliensis , also known as Para rubber tree, belonging to the Euphorbiaceae family, is indigenous to the Amazonian rainforest. Currently, it is cultivated worldwide for its latex, which is a major source of natural rubber, an important raw material for the commercial industry. More than 35,000 products are synthesized from natural rubber, including gloves, automobile and aircraft tires. Synthetic rubber cannot match natural rubber's exceptional properties, including tensile strength, elasticity, thermal and electrical insulation, and resistance to abrasion and wear and tear, as well as resilience (Kawahara et al. 2022 ). The market for natural rubber continues to expand due to rising demand from automotive sectors and growing interest in sustainable rubber sources. This demand supports the livelihood and economy of millions of smallholder farmers who contribute to rubber production. Thus, the production and productivity of natural rubber plays a decisive role in the economic growth and development of rubber-growing countries (Thangamalai 2021 ; Puskas et al. 2024 ). Globally, natural rubber is synthesized from the latex of H. brasiliensis . Latex is a milky white colloidal solution consisting of water (60%), cis-1,4-polyisoprene (35%), and non-isoprene molecules (5%) such as proteins, lipids, carbohydrates, and minerals (Bottier 2020 ). Specialized laticifer cells in the bark, leaves, and petioles of the rubber tree synthesize this valuable substance. Commercial latex extraction occurs through a process called tapping, which involves making a controlled incision through half the trunk's circumference at a 30° angle. This precise cut exposes latex vessels in the inner bark while avoiding damage to the cambium. Latex exudes from these incised laticifers and flows along the tapping panel, the channel created by this incision, into collection cups (Wang et al. 2023 ). While this method is designed to ensure sustainable production without harming the tree, repeated tapping can sometimes lead to physiological stress and health issues in the tree. One of the most significant challenges faced in this context is Tapping Panel Dryness (TPD), a condition where specific sections or entire tapping panel cease to produce latex despite regular tapping (Herlinawati et al. 2022 ). Unlike occasional dryness caused by improper tapping or temporary stress, TPD syndrome is more persistent due to irreversible cessation of latex flow and bark necrosis (Herlinawati et al. 2022 ). This condition substantially reduces a tree's productive lifespan through permanent blockage of latex biosynthesis and tissue death, severely impacting natural rubber production worldwide. TPD manifests as a complex syndrome rather than a disease with a single identifiable cause. Despite extensive research, its precise etiology remains elusive. While some studies suggest that the severity of TPD is dependent on the rubber tree clone (Tistama et al. 2019 ), the heritability of this trait has not been conclusively established by geneticists and is contested by conflicting reports of environmental stress and overexploitation (Herlinawati et al. 2022 ). The clustering pattern of TPD-affected trees observed in plantations and gradual spread of symptoms to adjacent trees over time (Abraham et al. 2013 ), raises the possibility of infectious agents associated with the development of the syndrome. Investigations into potential pathogenic agents, including viruses and viroids, have not provided definitive evidence of their involvement in TPD (Ramachandran et al. 2000 ; Zhao et al. 2023 ). Studies have reported bark abnormalities such as scaling, cracking, and necrotic streaking in association with TPD, yet comprehensive investigations have failed to implicate any fungal, bacterial, viral, or protozoan agents responsible for its onset (Pellegrin et al. 2004 ). This persistent knowledge gap necessitates alternative investigative approaches. Recent systematic investigation from the symptomatology, anatomical studies using confocal and scanning electron microscopy, followed by investigations on physiological changes in the TPD affected bark tissues, strongly suggested the possible presence of phytoplasmas (Philip et al. 2025 ). Hence, considering the fundamental role of prokaryotic communities in plant health and disease progression, this study was carried out to verify the phytoplasma presence and to determine whether composition of prokaryotic community in bark tissue differs between healthy and TPD-affected samples. 2. Materials and methods 2.1. Collection of samples and study site Bark samples were collected from a Tapping Panel Dryness (TPD)-affected plantation of clone RRII 430 (Fig. Ia) maintained by the Rubber Research Institute of India (RRII), Kottayam, Kerala, India (9.568689 N, 76.574478 E). The plantation had a TPD incidence rate of approximately 20%, and the trees were 15 years old, with no specific manurial treatments applied. Tapping was conducted using the S2d3 system, where the bark was cut spirally through half the circumference (S2) of the trunk once every three days (d3). Experimental trees exhibiting typical symptoms of TPD (Fig. Ib & Ic), such as complete blockage of latex flow, scaling, and peeling of bark, were selected as diseased samples. Healthy samples as control were collected from trees with intact bark, regular latex flow, and no visible symptoms of dryness (Fig. Id). Bark samples collected from healthy and TPD affected trees were subjected to anatomical and molecular investigation. 2.2 Anatomical investigation of healthy and TPD-affected bark samples Tangential longitudinal sections with an approximate thickness of 35 µm were prepared using a sliding microtome from the inner soft bark region of H. brasiliensis , a tissue rich in sieve tubes and latex vessels. The sections were then used to examine the presence of callose deposition, latex localization, putative phytoplasma detection, and ultrastructural alterations associated with tapping panel dryness (TPD) syndrome using a combination of histochemical stains, light microscopy, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). 2.2.1 Latex vessel staining using Oil Red O Oil red O based staining was used to identify latex-bearing cells in Hevea (Hamzah et al. 1988 ). Tangential longitudinal sections were treated with 60% isopropanol for 5 minutes followed by staining with 0.5% Oil Red O (w/v) (prepared in 100% isopropanol and diluted to 60% before use). Sections were incubated in the stain for 30 minutes, rinsed briefly in 60% isopropanol, and then mounted in 50% glycerol. Observations were made under a bright field light microscope (LEICA DM 2500 LED). 2.2.2 Callose staining with Congo Red To assess callose deposition in sieve tube elements, sections were stained with 0.1% (w/v) Congo Red (Hi Media, India) for 15 minutes in the dark at room temperature (Sivan et al. 2011 ). After two washes with distilled water, stained sections were mounted in distilled water and examined under a bright field light microscope (LEICA DM 2500 LED) 2.2.3 CLSM based detection of putative phytoplasma in the sieve tubes of bark sample Sections were fixed in 4% paraformaldehyde (w/v) in phosphate-buffered saline (PBS; pH 7.2) for 30 minutes at room temperature, washed thoroughly with PBS (3×5 min), and stained with 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Hi Media, India) for 20 minutes in the dark (Andrade and Arismendi 2013 ). After washing in PBS, samples were mounted in 50% glycerol and examined using a confocal laser scanning microscope (CLSM; Leica TCS-SP2, Rajiv Gandhi Centre for Biotechnology, Trivandrum, India) equipped with a UV laser (excitation at 358 nm, emission collected at 461 nm). Images were acquired using Leica Application Suite Advanced Fluorescence software (version 2.0). 2.2.4 Scanning electron microscopy (SEM) based ultrastructural detection of sieve tubes Tangential longitudinal bark sections were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 hours at 4°C, post-fixed in 1% osmium tetroxide for 1 hour, and dehydrated in an ascending ethanol series (30% to 100%) (Philip et al. 2025 ). Samples were critical-point dried using a BAL-TEC CPD 030 Critical Point Dryer ( Rajiv Gandhi Centre for Biotechnology, Trivandrum, India ) and mounted on aluminium stubs using double-sided carbon tape. Specimens were sputter-coated with gold in a BAL-TEC SCD 005 sputter coater and examined using a JEOL JSM-6390LV scanning electron microscope operated at 10 and 15 kV. 2.3 Metagenomic analysis of prokaryotic communities in healthy and TPD-affected bark samples Total genomic DNA were isolated from bark tissues of healthy and TPD-affected trees and used for 16S rRNA gene-based library preparation and high-throughput sequencing to profile the associated microbial communities. For each condition (healthy and TPD-affected), two trees were selected as biological replicates. 2.3.1 DNA extraction from the bark samples of healthy and TPD affected trees Bark samples from multiple portions of the trunk were processed by first removing the outer cortex and grinding the remaining tissue in liquid nitrogen to obtain a fine powder. DNA extraction was performed using a slightly modified version of the protocol described by (Doyle and Doyle 1987 ). Approximately 2 g of ground tissue was mixed with preheated CTAB buffer (2% CTAB, 1% polyvinyl pyrrolidone, 1.4 M sodium chloride, 50 mM EDTA, 100 mM Tris-HCl at pH 7.8, and 0.2% β-mercaptoethanol). The lysate was incubated at 65°C for 30 minutes, followed by the addition of an equal volume of chloroform:isoamyl alcohol (24:1) and centrifugation at 15,000g for 25 seconds. The supernatant was carefully collected, treated with 4 µl of RNase, and incubated at 37°C for 30 minutes. DNA was precipitated by adding two-third volume of ice-cold isopropanol. The mixture was centrifuged at 15,000g for 25 seconds to recover the pellet. The resulting pellet was washed with 70% ethanol, air-dried, and dissolved in Milli-Q water. The quality and quantity of the extracted DNA were assessed using agarose gel electrophoresis and Invitrogen Qubit 4 Flurometer for suitability in PCR-based amplification of the 16S rRNA gene. Metagenome sequencing, library preparation and bioinformatic analysis were done with the help of commercial sequencing service providers Eurofins Genomics India, Bengaluru and CloneGtyping, Bengaluru, both from India. 2.3.2 Preparation of 2 x 300 MiSeq library The amplicon library was prepared using the Nextera XT Index Kit. Primers targeting the bacterial-specific region were designed by Eurofins Genomics Lab, India (forward primer: 5′ GCCTACGGGNGGCWGCAG 3′ and reverse primer: 5′ ACTACHVGGGTATCTAATCC 3′). Quality-checked (QC) amplicons with Illumina adapters were further amplified using i5 and i7 primers (Illumina 2013 ), which incorporate multiplexing index sequences and common adapters (P5 and P7) (Illumina 2013 ) essential for cluster generation. The amplicon library was purified using AMPure XP beads and quantified with a Qubit Fluorometer. Amplified libraries underwent fragment analysis on the 4200 TapeStation system (Agilent Technologies) using D1000 ScreenTape, following the manufacturer's protocol. Libraries passing QC were sequenced on the MiSeq platform. The resulting sequences from this study have been deposited in the Sequence Read Archive (SRA) under BioProject ID: PRJNA1203434. 2.3.3 Sequence processing, statistical analysis and functional prediction The raw data quality was assessed using the FastQC tool (S. Andrews 2010 ). The reads were imported into the QIIME2 environment (Bolyen et al. 2019 ) via the qiime tools import function. After importing, the reads were merged to form a consensus sequence, and a Phred score of 20 was applied as the quality filtering parameter for downstream analyses. Subsequent quality control steps included denoising and dereplication using the qiime vsearch dereplicate-sequences function (Rognes et al. 2016 ), which collapsed identical sequences into unique representative sequences. Chimeric sequences were identified and removed using the qiime vsearch uchime-denovo function (Edgar et al. 2011 ). Operational Taxonomic Units (OTUs) were clustered de novo at 97% sequence similarity using the qiime vsearch cluster-features-de-novo command, grouping similar sequences into OTUs that represent distinct bacterial taxa. The OTUs were classified using the SILVA 138.1 database. The qiime feature-table summarize function was then used to generate the feature/OTU table. For further analysis, the Microeco package (Liu et al. 2020 ) ( https://github.com/ChiLiubio/microeco ) was utilized. The relative abundance of the top 10 phyla and 12 genera was calculated and visualized using boxplots. Alpha diversity metrics, including observed species, Chao1, ACE, Shannon, Simpson, and Inverse Simpson indices, were computed, with significant variations determined by a t-test (p < 0.05). To predict the functional potential of the microbial communities, PICRUSt2 (Douglas et al. 2020 ) was employed. Representative sequences in FASTA format and the OTU table (BIOME/TSV format) were exported from QIIME2 for input. The pipeline began by placing Amplicon Sequence Variants (ASVs) onto a reference phylogeny using Hidden-State Prediction (HSP). Gene family abundances, including KEGG Orthologs (KO) and EC numbers, were predicted based on the phylogenetic positioning of the ASVs, and pathway-level predictions were made using MinPath. PICRUSt2 further normalized these predictions to account for variations in 16S rRNA gene copy numbers. The predicted gene families and metabolic pathways were analyzed and visualized using STAMP (Parks et al. 2014 ), providing insights into the functional potential of the microbial communities. 3. Results 3.1 Anatomical differences in the healthy and TPD-affected bark samples of H. brasiliensis To understand the anatomical changes associated with Tapping Panel Dryness (TPD) in H. brasiliensis , comparative observations were made between healthy and TPD-affected bark tissues, particularly focusing on sieve tubes, latex vessels, and the presence of intracellular phytoplasma-like bodies. The following subsections detail the findings from each method. 3.1.1 Visualization of latex vessel organization in healthy and TPD-affected bark samples In healthy bark sections, laticiferous tissues appeared as well-defined, regularly aligned structures, with uniform red staining (Fig. II a). The Oil Red O selectively stained lipid-rich latex vesicles, which were densely packed within the laticifers, indicating normal rubber biosynthesis and storage activity. In contrast, bark tissues from TPD-affected trees (Fig. II b) exhibited noticeable reduction in Oil Red O staining intensity with deformed latex vessel distribution. Dark greyish discoloration was observed in ray parenchyma cells and latex vessels indicating impaired rubber biosynthetic activity or altered metabolic processes associated with the onset of TPD. 3.1.2 Callose deposition in sieve tubes of healthy and TPD-affected bark samples Sections stained with Congo red were observed for callose deposition. Marked differences were observed between healthy and TPD-affected samples stained with Congo red under bright-field light microscopy. At lower magnification (10X), prominent red staining was localized in the sieve plates of TPD-affected sample (Fig. III). At higher magnification (40X), discrete red bands were observed occluding sieve pores, indicating dense callose deposits across multiple sieve elements (Fig. IV). These occlusions were largely absent or minimal in healthy samples, where sieve plates appeared clear and structurally intact (Fig. IV), indicating free axial and radial transport in the sieve tube. 3.1.3 DAPI fluorescence microscopy reveals nuclear material in sieve tubes of TPD-affected sample Bark sections stained with DAPI, were examined to detect the presence of nuclear material in sieve elements. In healthy bark tissues, no fluorescence signal was observed, indicating the absence of extranuclear DNA in sieve tube elements (Fig. V). In contrast, sections from TPD-affected samples showed prominent DAPI fluorescence localized within sieve elements. These bright, punctate signals suggest the presence of DNA-containing bodies, or microbes potentially corresponding to phytoplasma. 3.1.4 Scanning Electron Microscopy (SEM) of sieve tube of TPD-affected bark sample SEM imaging of TPD-affected bark sections, at 10 and 15 kV revealed the presence of pleomorphic bodies distributed in the sieve tube at a size ranging from 372 to 991 nm (Fig. VI). 3.2 Composition, diversity and functional potential of prokaryotic communities in healthy and TPD-affected samples Metagenomic analysis of bark tissues from healthy and TPD-affected H. brasiliensis trees provided insights into the prokaryotic community structure, diversity, and predicted functional potential, with results described in the following subsections. 3.2.1 Quantitative and qualitative analysis of DNA and amplicon libraries from healthy and TPD-affected bark samples The DNA yield varied across samples, with healthy and TPD affected bark yielded an average of 128.1 and 59.8 ng/µL, respectively. The purity of all DNA samples was within an acceptable range (260/280 ratio ~ 1.8), ensuring their suitability for amplicon library preparation. Amplification of 16S rRNA generated high quality amplicons of an average size 595 bp which was standardized to 19 ng/µL for sequencing. 3.2.2 Sequencing overview and taxonomic profiling of healthy and TPD-affected bark microbiomes Sequencing of amplicon libraries generated a substantial number of raw reads, which were subsequently quality filtered to obtain high-confidence sequences of ~ 301 bp, for taxonomic analysis. In healthy samples, a total of 1,52,547 quality (HQ) reads yielded 2,111 operational taxonomic units (OTUs). The classification of these sequences identified 30 phyla, 93 class, 174 order, 310 family and 500 genera. In contrast, TPD-affected samples exhibited a total of 1,69,807 reads with 2,224 OTUs identified. Their classification revealed a larger microbial diversity in TPD-affected samples, comprising 30 phyla, 92 class, 175 order, 313 family and 505 genera. 3.2.3 Comparative analysis of bacterial communities in healthy and TPD affected samples Microbial community composition showed no significant differences between healthy and TPD-affected bark samples at any taxonomic level (p > 0.05). Bacteria and Archaea were present in similar proportions, with Proteobacteria and Actinobacteriota as dominant phyla in both conditions. Differences in relative abundances across the major taxa were not statistically significant (Fig. VII, S1&S2). However, a few prokaryotes namely, Comamonas, Niastella , Phytoplasma, Salinibacter , and Wolbachia were detected exclusively in TPD-affected samples, albeit at very low abundances (< 0.01%) (Table. I). 3.2.4 Diversity analysis and richness estimates Alpha diversity of the samples was analyzed using various diversity indices like Simpson, Chao, Shannon-Wiener, Abundance-based Coverage Estimator (ACE) index, Inverse simpson (Fig. VIII). Species richness was analyzed using Simpson diversity index and Shannon index. Richness of rare species was estimated by Chao 1 and ACE index. Diseased samples exhibited higher variation across all indices as indicated by larger spread in box plots. Consistently lower indices in healthy samples suggest uniformity of microbial community. However, statistically these differences between healthy and infected samples resulted not significant (p > 0.05). 3.2.5 Predicted pathways from 16S rRNA A total of 7418 PICRUSt2 predicted KEGG orthologs (enzymes) were collapsed to 426 KEGG pathways. The analysis revealed significant alterations in various metabolic pathways between healthy and infected individuals (Fig. IX). In a broader way of categorization in healthy samples, pathways involving amino acid biosynthesis, glycan biosynthesis and metabolism and carbohydrate metabolism resulted highly enriched. Among them, superpathway of methylglyoxal degradation (glycan biosynthesis) and Entner-Doudoroff pathway III (carbohydrate metabolism) were significantly abundant (q < 1 e − 15 ). Among the top 25 differentially abundant pathways, the superpathway of bacteriochlorophyll a biosynthesis and isopropanol biosynthesis exhibited the most pronounced increase in the infected group, with a significant difference between each other (q < 1 e − 15 ). Additionally, pathways related to formaldehyde assimilation (serine pathway), chlorosalicylate degradation and protocatechuate degradation also showed significant overrepresentation in infected individuals. This suggests enhanced microbial biodegradation of xenobiotics or host metabolites. 4. Discussion This study combined anatomical, cytological, metagenomic, and molecular approaches to investigate potential biotic associations with TPD, with a specific focus on vascular tissue integrity and microbial community dynamics. The multidisciplinary findings of the study propose a unified model of TPD pathogenesis and highlights the significance of phytoplasma association in TPD-affected trees. Latex flow in H. brasiliensis is highly dependent on the structural and functional integrity of laticifers and their associated phloem tissues (De Faÿ 2011 ). Latex vessels stained with Oil red O in the healthy samples appeared structurally intact. As Oil red O has strong affinity for neutral lipids, uniform and intense red staining in healthy samples, indicate abundant and well-preserved lipids in the laticifers (Hamzah et al. 1988 ). Congo red staining of tangential bark sections from healthy H. brasiliensis revealed well-organized sieve tube elements with clearly visible perforated sieve plates. The sieve plates, which form the interface between adjacent sieve elements, appeared open and unobstructed, allowing unimpeded transport of photosynthates and signaling molecules. Latex vessel absorbs nutrients from neighboring sieve tubes for latex biosynthesis (Sivan et al. 2011 ). The absence of callose or phenolic deposition in healthy sample suggests that the phloem is physiologically active and free of stress-induced blockages, maintaining uninterrupted nutrient supply to neighboring tissues, including latex vessels (De Faÿ 2011 ). Hence, disruption in sieve tube function directly impacts the metabolic activity of adjacent laticifers (Sivan et al. 2011 ). Congo Red, a diazo dye with specific affinity for β-1,3-glucans (Piccinini et al. 2024 ), revealed prominent callose deposition and blockage in the sieve plates of TPD-affected bark. Such blockages impair both radial and axial transport in the phloem, leading to nutrient deprivation in latex vessels. This, in turn, disrupts rubber biosynthesis and promotes metabolic stress. Anatomical symptoms such as reduced lipid staining, deformation of latex vessels, and darkened cell contents in TPD-affected samples are consistent with previous studies linking nutrient starvation and oxidative damage in latex vessels due to sieve tube dysfunction (Sivan et al. 2011 ; Zhang et al. 2016 ). Stress in sieve tubes, whether from biotic or abiotic origins, can thus severely impact latex production. DAPI staining in confocal laser scanning microscopy (CLSM) revealed fluorescence exclusively in TPD-affected samples. As sieve tubes are anucleate and DAPI binds to AT-rich DNA, this fluorescence strongly suggests the presence of microbial DNA, most plausibly phytoplasma, known to inhabit sieve tubes and possess AT-rich genomes (Andrade and Arismendi 2013 ). SEM further confirmed the presence of pleomorphic bodies (319–990 nm) resembling phytoplasmas in TPD-affected samples. These findings align with previous report of association of phytoplasma with TPD-affected trees of H. brasiliensis clone RRII 105 (Philip et al. 2025 ). 16S rRNA based metagenomic analysis was done to understand the overall prokaryotic composition and diversity in healthy and TPD-affected sample. The bacterial community composition remains consistent across healthy and TPD-affected samples, with no significant shifts in dominant microbial groups at higher taxonomic levels. At the kingdom level, both healthy and TPD-affected samples exhibited similar proportions of Bacteria and Archaea. Various other plants like rice, maize and Scots pine also have reported to be associated with several groups of Archaea in their phytobiome (Jung et al. 2020 ). Proteobacteria and Actinobacteria were the most abundant bacterial phyla in both samples. These major phyla are commonly present in almost all ecosystems. Acidobacteria, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes are dominant phyla reported from bark of trees like avocado and red oak (Aguirre-von-Wobeser 2020 ; Hudson et al. 2023 ). The microbial community composition at all taxa remains largely similar in healthy and TPD-affected samples. This suggests that the overall microbiome composition may not be heavily impacted by the disease, or that the disease is not directly associated with large-scale microbial shifts detectable through 16S rRNA amplicon sequencing. Amplicon sequencing, which targets conserved regions ( e.g ., 16S rRNA), may not be sensitive enough to detect functional differences or low-abundance microbes that could play a critical role in disease development (Rizal et al. 2020 ). Of particular interest is the presence of phytoplasma, with a very low concentration exclusively detected in TPD affected samples. Although detected at extremely low concentrations, phytoplasmas possess the potential to alter host plant metabolism profoundly. Previous studies have shown that concentrations as low as 370 to 34,000 cells per g. of tissue is associated with symptoms of witches’ broom in apple trees, with nested PCR detecting as few as 4 to 340 cells in reaction mixtures (Berges et al. 2000 ). Diseases like, elm yellows, peach X disease, apple proliferation, arecanut lethal yellowing and grapevine bois noir are some of the examples of disease associated with phytoplasma presence in perennial crops with serious economic impact (Wei et al. 2022 ; Wang et al. 2024 ). Furthermore, the minimal concentration of phytoplasmas might explain their low abundance in metagenomic sequencing data, while a possible role in pathogenesis emphasizes their ability to induce disease without significantly altering the microbial diversity or composition in the host microbiome. Recent studies indicate that not all infections lead to major changes in microbial composition. In some cases, like strawberry with signs of plant stress or pathogens, the microbiome showed limited variation between healthy and diseased plants, with only a few taxa displaying noticeable shifts. This suggests that even under pathogenic stress, a stable core microbiome may be retained (Hassani et al. 2023 ). Phytoplasmas are prokaryotes lacking rigid cell wall. However, their genome size is greatly reduced as a result of reductive evolution from Gram-positive bacteria belonging to Firmicutes. Lack of several genes for basic metabolic pathways and dependence on sucrose as the main source of carbon made them an obligate plant pathogen inhabiting phloem sieve tubes (Oshima 2021 ). Given that sieve tubes are rich in sucrose, a precursor for rubber biosynthesis, their blockage due to thick callose and P-protein deposition of TPD-affected trees hint the potential role of phytoplasmas in inducing the syndrome (Sivan et al. 2011 ; Buxa-Kleeberg et al. 2015 ). Plant defense mechanisms, such as callose deposition and aggregation of P-protein filaments in sieve plates, are reported in plants infected by phytoplasmas (Gallinger and Gross 2020 ; Wei et al. 2022 ). Nutrient deprivation, coupled with stress-induced defense signaling, could trigger hypersensitive responses and programmed cell death in laticifers, ultimately disrupting latex biosynthesis (Zimmermann et al. 2015 ; Meisrimler et al. 2021 ). By linking phytoplasma detection to characteristic symptoms like callose deposition, sieve tube blockage, and laticifer shrinkage, highlights its potential as a critical pathogen underlying TPD, even at low concentrations. Analysis revealed that there is no significant variation in microbial diversity indices for healthy and TPD-affected samples. This implies that TPD may not be heavily impacted by the microbial richness and evenness. Amplicon metagenomics often capture overall community composition wherein low titre and localized microbes' effect on richness, evenness and microbial community structure gets unaffected (Cena et al. 2021 ). Well supported by the information on symptomatology, pattern of spread, and anatomical changes which are obtained through other experiments in this study, the data on metagenomics suggests or rather detects biologically meaningful trends (Dahlberg et al. 2023 ) indicating the presence of phytoplasmas in TPD. Despite the similar microbial diversity and composition, there is a significant difference in the metabolic pathways enriched in healthy and TPD affected samples. This could possibly be due to calculation of diversity metrics at a broad taxonomic level ( e.g ., genus or species), and they may not be sensitive enough to detect strain-level or functional differences that are important for pathway prediction. However, the metabolic pathways identified in this study were predicted using PICRUSt, which infers functional potential based on more detailed taxonomic resolution or phylogenetic relationships (Sun et al. 2020 ; Toole et al. 2021 ). While these results provide valuable insights into potential microbial metabolic shifts, they do not represent direct biochemical activity. Pathways involved in phenolic compound degradation, such as methylgallate degradation, gallate degradation, and chlorosalicylate degradation, were significantly enriched in diseased samples. These compounds are known to play a role in plant defense, and their increased degradation may represent a counter-defense mechanism by pathogens to neutralize plant defenses and establish infections (Zhang et al. 2020 , 2022 ; Soal et al. 2022 ). Phytoplasma infections are known to interfere with host secondary metabolism, particularly phenylpropanoid and salicylic acid pathway s , which could indirectly influence microbial functional dynamics (Pradit et al. 2019 ; Bauters et al. 2021 ; Dermastia et al. 2023 ). 5. Conclusion This study presents a multidisciplinary investigation into the possible biotic factors associated with TPD in H. brasiliensis . While there are no major shifts in the dominant microbial groups between healthy and TPD-affected bark, the functional insights from pathway analysis, particularly the enrichment of biodegradative pathways in TPD-affected samples, open avenues for further exploration into the role of microbial metabolism in disease dynamics and possible microbial interactions with host metabolites. Lack of amplification of rare taxa is one of the limitations of amplicon based metagenomic sequencing and hence make it difficult to draw a conclusion on the abundance of microbes. Anatomical and cytological alterations in phloem tissues, alongside metagenomic and PCR based evidences, suggest the presence of a divergent phytoplasma strain in the TPD-affected samples. Although at low concentration, the detection of phytoplasma sequences could be significant, suggesting their potential pathogenic role in TPD. Further studies using targeted and deeper sequencing techniques will elucidate its potential contribution to disease development. Current investigation provides preliminary insights into the biotic complexity of TPD and lay groundwork for future confirmatory studies. Declarations Funding This work was supported in part by Kerala Agricultural University, Thrissur and Rubber research Institute of India, Kottayam. Acknowledgements The authors express their sincere gratitude to Rubber Research Institute of India for providing laboratory facilities and also acknowledge the valuable assistance of my colleagues Vineeth V. K, Shilpa Babu, Swathi S. J and Rahul Raj for the successful completion of the current research work. The authors are also grateful to Eurofins Genomics India Pvt. Ltd, Bengaluru and CloneGtyping, Bengaluru for metagenome sequencing and bioinformatics analysis. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work the authors used free version of Claude in order to improve the writing. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Author Contribution A.T. conducted experiment and wrote manuscript text. S.K.P. and S.P. structured the research work and reviewed the manuscript. All other authors reviewed manuscript References Abraham A, Philip S, Kuruvilla Jacob C, Jayachandran K (2013) Novel bacterial endophytes from Hevea brasiliensis as biocontrol agent against Phytophthora leaf fall disease. BioControl 58:675–684. https://doi.org/10.1007/s10526-013-9516-0 Aguirre-von-Wobeser E (2020) Functional metagenomics of bark microbial communities from avocado trees ( Persea americana Mill.) reveals potential for bacterial primary productivity. bioRxiv. https://doi.org/10.1101/2020.09.05.284570 Alessio FI, Bongiorno VA, Marcone C et al (2025) Genetic Diversity in Phytoplasmas from X-Disease Group Based in Analysis of idpA and imp Genes. Microorganisms 13:1–14. https://doi.org/10.3390/microorganisms13051170 Andrade NM, Arismendi NL (2013) DAPI Staining and Fluorescence Microscopy Techniques for Phytoplasmas. In: Dickinson M, Hodgetts J (eds) Phytoplasma: Methods and Protocols. Humana Press, Totowa, NJ, pp 115–121 Bauters L, Stojilković B, Gheysen G (2021) Pathogens pulling the strings: Effectors manipulating salicylic acid and phenylpropanoid biosynthesis in plants. Mol Plant Pathol 22:1436–1448. https://doi.org/10.1111/mpp.13123 Berges R, Rott M, Seemuller E (2000) Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction. Phytopathology 90:1145–1152. https://doi.org/10.1094/PHYTO.2000.90.10.1145 Bolyen E, Rideout JR, Dillon MR et al (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37:852–857. https://doi.org/10.1038/s41587-019-0209-9 Bottier C (2020) Biochemical composition of Hevea brasiliensis latex: A focus on the protein, lipid, carbohydrate and mineral contents. Adv Bot Res 93:201–237. https://doi.org/10.1016/bs.abr.2019.11.003 Buxa-Kleeberg S, Degola F, Polizzotto R et al (2015) Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements. Front Plant Sci 6:650 Cena JA de, Zhang J, Deng D et al (2021) Low-Abundant Microorganisms: The Human Microbiome’s Dark Matter, a Scoping Review. Front Cell Infect Microbiol 11:. https://doi.org/10.3389/fcimb.2021.689197 Dahlberg J, Pelve E, Dicksved J (2023) Similarity in milk microbiota in replicates. Microbiologyopen 12:1–9. https://doi.org/10.1002/mbo3.1383 De Faÿ E (2011) Histo- and cytopathology of trunk phloem necrosis, a form of rubber tree ( Hevea brasiliensis Muell. Arg.) tapping panel dryness. Aust J Bot 59:563–574. https://doi.org/10.1071/BT11070 Dermastia M, Tomaž Š, Strah R et al (2023) Candidate pathogenicity factor/effector proteins of ‘ Candidatus Phytoplasma solani’ modulate plant carbohydrate metabolism, accelerate the ascorbate–glutathione cycle, and induce autophagosomes. Front Plant Sci 14:1–16. https://doi.org/10.3389/fpls.2023.1232367 Douglas GM, Maffei VJ, Zaneveld JR et al (2020) PICRUSt2 for prediction of metagenome functions. Nat. Biotechnol. 38:685–688 Doyle . J.J., Doyle . J.L. (1987) a Rapid Dna Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochem. Bull. 19:11–15 Edgar RC, Haas BJ, Clemente JC et al (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200. https://doi.org/10.1093/bioinformatics/btr381 Felsenstein J (1985) Confidence Limits on Phylogenies: an Approach Using the Bootstrap. Evolution (N Y) 39:783–791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x Gallinger J, Gross J (2020) Phloem Metabolites of Prunus Sp. Rather than Infection with Candidatus Phytoplasma Prunorum Influence Feeding Behavior of Cacopsylla pruni Nymphs. J Chem Ecol 46:756–770. https://doi.org/10.1007/s10886-020-01148-8 Hamzah S, Gomez JB, Ho LH (1988) a Refinement of the Staining Techniques for Hevea Latex Vessels. J Nat Rubber Res 3:163–166 Hassani MA, Gonzalez O, Hunter SS, et al (2023) Microbiome Network Connectivity and Composition Linked to Disease Resistance in Strawberry Plants. Phytobiomes J 298–311. https://doi.org/10.1094/pbiomes-10-22-0069-r Herlinawati E, Montoro P, Ismawanto S et al (2022) Dynamic analysis of Tapping Panel Dryness in Hevea brasiliensis reveals new insights on this physiological syndrome affecting latex production. Heliyon 8:e10920. https://doi.org/10.1016/j.heliyon.2022.e10920 Hudson JE, Levia DF, Yoshimura KM et al (2023) changes in bark surface phyla. 11:1–16 Illumina (2013) 16S Metagenomic Sequencing Library. Illumina.com 1–28 Jung J, Kim JS, Taffner J et al (2020) Archaea, tiny helpers of land plants. Comput Struct Biotechnol J 18:2494–2500. https://doi.org/10.1016/j.csbj.2020.09.005 Kawahara S, Nishioka H, Yamano M, Yamamoto Y (2022) Synthetic Rubber with the Tensile Strength of Natural Rubber. ACS Appl Polym Mater 4:2323–2328. https://doi.org/10.1021/acsapm.1c01508 Kim M, Oh H-S, Park S-C, Chun J (2014) Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64:346–351. https://doi.org/10.1099/ijs.0.059774-0 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120. https://doi.org/10.1007/BF01731581 Liu C, Cui Y, Li X, Yao M (2020) microeco : An R package for data mining in microbial community ecology. FEMS Microbiol Ecol 97:. https://doi.org/10.1093/femsec/fiaa255 Meisrimler CN, Allan C, Eccersall S, Morris RJ (2021) Interior design: how plant pathogens optimize their living conditions. New Phytol 229:2514–2524. https://doi.org/10.1111/nph.17024 Oshima K (2021) Molecular biological study on the survival strategy of phytoplasma. J Gen Plant Pathol 87:. https://doi.org/10.1007/s10327-021-01027-4 Oshima K, Kakizawa S, Nishigawa H et al (2004) Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nat Genet 36:27–29. https://doi.org/10.1038/ng1277 Parks DH, Tyson GW, Hugenholtz P, Beiko RG (2014) STAMP: statistical analysis of taxonomic and functional profiles. Bioinformatics 30:3123–3124. https://doi.org/10.1093/bioinformatics/btu494 Pellegrin, F. Nandris, D. and Chrestin H (2004) Rubber Tree ( Hevea brasiliensis ) Bark Necrosis Syndrome I: Still No Evidence of a Biotic Causal Agent. Plant Dis 88:1,046.3-1,046.3. https://doi.org/http://dx.doi.org/10.1094/PDIS.2004.88.9.1046C Piccinini L, Nirina Ramamonjy F, Ursache R (2024) Imaging plant cell walls using fluorescent stains: The beauty is in the details. J Microsc 295:102–120. https://doi.org/10.1111/jmi.13289 Pradit N, Rodriguez-Saona C, Kawash J, Polashock J (2019) Phytoplasma Infection Influences Gene Expression in American Cranberry. Front Ecol Evol 7:1–14. https://doi.org/10.3389/fevo.2019.00178 Puskas JE, Cornish K, Kenzhe-Karim B et al (2024) Natural rubber – Increasing diversity of an irreplaceable renewable resource. Heliyon 10:e25123. https://doi.org/https://doi.org/10.1016/j.heliyon.2024.e25123 Ramachandran P, Mathur S, Francis L et al (2000) Evidence for Association of a Viroid with Tapping Panel Dryness Syndrome of Rubber (Hevea brasiliensis). Plant Dis 84:1155. https://doi.org/10.1094/PDIS.2000.84.10.1155C Rizal NSM, Neoh HM, Ramli R et al (2020) Advantages and limitations of 16S rRNA next-generation sequencing for pathogen identification in the diagnostic microbiology laboratory: perspectives from a middle-income country. Diagnostics 10:. https://doi.org/10.3390/diagnostics10100816 Rognes T, Flouri T, Nichols B et al (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. https://doi.org/10.7717/peerj.2584 Andrews S (2010) FastQC: a quality control tool for high throughput sequence data Philip S, Tom A, Prem E, Puramerimadathil R, Sajeed RK (2025) Association of phytoplasmas with tapping panel dryness syndrome of Hevea brasiliensis : a pathological perspective to unresolved mystey? Phytopathogenic Mollicutes 15:63–64 Sivan P, Thomas V, Rao K, Krishnakumar R (2011) Definitive Callose Deposition in Tapping Panel Dryness Affected Bark of Hevea brasiliensis . J Sustain For 30:329–342. https://doi.org/10.1080/10549811.2011.532032 Smart CD, Schneider B, Blomquist CL et al (1996) Phytoplasma-Specific PCR Primers Based on Sequences of the 16S-23S rRNA Spacer Region. 62:2988–2993 Soal NC, Coetzee MPA, van der Nest MA et al (2022) Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae. G3 Genes, Genomes, Genet 12:. https://doi.org/10.1093/g3journal/jkac008 Sun S, Jones RB, Fodor AA (2020) Inference-based accuracy of metagenome prediction tools varies across sample types and functional categories. Microbiome 8:1–9. https://doi.org/10.1186/s40168-020-00815-y Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 38:3022–3027. https://doi.org/10.1093/molbev/msab120 Thangamalai A (2021) Growth and Prospects of Natural and Synthetic Rubber Production and Consumption in India. Rev Gestão Inovação e Tecnol 11:2019–2032. https://doi.org/10.47059/revistageintec.v11i4.2251 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. https://doi.org/10.1093/nar/22.22.4673 Tistama R, Mawaddah PAS, Ade-Fipriani L, Junaidi (2019) Physiological status of high and low metabolism Hevea clones in the difference stage of tapping panel dryness. Biodiversitas 20:267–273. https://doi.org/10.13057/biodiv/d200143 Toole DR, Zhao J, Martens-Habbena W, Strauss SL (2021) Bacterial functional prediction tools detect but underestimate metabolic diversity compared to shotgun metagenomics in southwest Florida soils. Appl Soil Ecol 168:104129. https://doi.org/10.1016/j.apsoil.2021.104129 Wang L, Huang C, Li T et al (2023) An Optimization Study on a Novel Mechanical Rubber Tree Tapping Mechanism and Technology. Forests 14:1–25. https://doi.org/10.3390/f14122421 Wang R, Bai B, Li D et al (2024) Phytoplasma: A plant pathogen that cannot be ignored in agricultural production—Research progress and outlook. Mol Plant Pathol 25:1–19. https://doi.org/10.1111/mpp.13437 Wei W, Inaba J, Zhao Y et al (2022) Phytoplasma Infection Blocks Starch Breakdown and Triggers Chloroplast Degradation, Leading to Premature Leaf Senescence, Sucrose Reallocation, and Spatiotemporal Redistribution of Phytohormones. Int J Mol Sci 23:. https://doi.org/10.3390/ijms23031810 Zhang J, Coaker G, Zhou JM, Dong X (2020) Plant Immune Mechanisms: From Reductionistic to Holistic Points of View. Mol Plant 13:1358–1378. https://doi.org/10.1016/j.molp.2020.09.007 Zhang Q, Li M, Yang G et al (2022) Protocatechuic acid, ferulic acid and relevant defense enzymes correlate closely with walnut resistance to Xanthomonas arboricola pv. juglandis. BMC Plant Biol 22:1–14. https://doi.org/10.1186/s12870-022-03997-9 Zhang Y, Leclercq J, Montoro P (2016) Reactive oxygen species in Hevea brasiliensis latex and relevance to Tapping Panel Dryness. 2:261–269. https://doi.org/10.1093/treephys/tpw106 Zhao R, Su X, Yu F et al (2023) Identification and characterization of two closely related virga-like viruses latently infecting rubber trees ( Hevea brasiliensis ). Front Microbiol 14:1286369. https://doi.org/10.3389/fmicb.2023.1286369 Zimmermann MR, Schneider B, Mithöfer A et al (2015) Implications of Candidatus Phytoplasma mali infection on phloem function of apple trees. J Endocytobiosis Cell Res 26:67–75 Additional Declarations No competing interests reported. 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09:50:24","extension":"html","order_by":46,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":171478,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/40d696f5f051383a6b180f19.html"},{"id":98054936,"identity":"b19d179d-42e0-438b-8086-fb935d437ec6","added_by":"auto","created_at":"2025-12-12 09:50:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":670617,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Rubber plantation (b) Dried panel (c) TPD-affected rubber tree (d) Healthy rubber tree\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/29ac019d3c10a199265f059e.png"},{"id":98054937,"identity":"89abfba7-74ef-4403-8fff-903b2f7f9204","added_by":"auto","created_at":"2025-12-12 09:50:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":528887,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Latex vessels in healthy sample (b) Latex vessels in TPD-affected sample; (10X magnification)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/03cf28a9a7b58510faf2cd7a.png"},{"id":98428608,"identity":"dfe304de-8233-46ac-8125-5f13e499e560","added_by":"auto","created_at":"2025-12-17 16:42:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":529363,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Tangential longitudinal section of inner soft bark region stained with Congo red (10X magnification)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/be7a2ada44e074b7cbf6c62f.png"},{"id":98428586,"identity":"23bf2320-52b1-4705-9a2b-822209639b29","added_by":"auto","created_at":"2025-12-17 16:42:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":487927,"visible":true,"origin":"","legend":"\u003cp\u003eSieve plates (40X magnification)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/5cbc390595cb95fa7d3a0aac.png"},{"id":98428613,"identity":"e3e55cfd-55e6-4f46-9375-36a3c2a6d596","added_by":"auto","created_at":"2025-12-17 16:42:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":233641,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning micrograph of sieve tubes\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/fdd2c2056787f43901d2ea61.png"},{"id":98054949,"identity":"76dea385-d462-4a60-bb0d-d708937b6fc6","added_by":"auto","created_at":"2025-12-12 09:50:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":326730,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrograph of sieve tubes of TPD affected bark\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/7bded6cf8a391c588160a6a8.png"},{"id":98427602,"identity":"56e4c423-99ae-43ef-9e1d-0f5a21e07abd","added_by":"auto","created_at":"2025-12-17 16:40:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":117156,"visible":true,"origin":"","legend":"\u003cp\u003e(a)\u003cstrong\u003e \u003c/strong\u003eAbundance of microbes at phylum level (b) Abundance of microbes at family level (c) Abundance of microbes at genus level\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/530e8d1b022cff3db62ee571.png"},{"id":98054945,"identity":"26dd65e7-a69d-4d10-b5b7-8ef1ea7ec6bd","added_by":"auto","created_at":"2025-12-12 09:50:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":48352,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Simpson index (b) Chao index (c) Shannon-Wiener index (d) ACE index (e) Inverse Simpson index\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/2368fd35d9faf3d4e9feeec0.png"},{"id":98054951,"identity":"fbc3f900-4831-4472-a15b-374117afabcc","added_by":"auto","created_at":"2025-12-12 09:50:24","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":106354,"visible":true,"origin":"","legend":"\u003cp\u003e16S rRNA based predicted metabolic pathways\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/f45f2138fa078a2ee3da7446.png"},{"id":106809175,"identity":"6606b20a-ec92-4fe1-adb4-6fc6947d1ac0","added_by":"auto","created_at":"2026-04-13 16:07:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4491525,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8276740/v1/5a1a9c52-71eb-4a85-afb4-effc843f66dd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploratory profiling of microbial communities associated with tapping panel dryness in Hevea brasiliensis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eHevea brasiliensis\u003c/em\u003e, also known as Para rubber tree, belonging to the Euphorbiaceae family, is indigenous to the Amazonian rainforest. Currently, it is cultivated worldwide for its latex, which is a major source of natural rubber, an important raw material for the commercial industry. More than 35,000 products are synthesized from natural rubber, including gloves, automobile and aircraft tires.\u003c/p\u003e\u003cp\u003eSynthetic rubber cannot match natural rubber's exceptional properties, including tensile strength, elasticity, thermal and electrical insulation, and resistance to abrasion and wear and tear, as well as resilience (Kawahara et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The market for natural rubber continues to expand due to rising demand from automotive sectors and growing interest in sustainable rubber sources. This demand supports the livelihood and economy of millions of smallholder farmers who contribute to rubber production. Thus, the production and productivity of natural rubber plays a decisive role in the economic growth and development of rubber-growing countries (Thangamalai \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Puskas et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGlobally, natural rubber is synthesized from the latex of \u003cem\u003eH. brasiliensis\u003c/em\u003e. Latex is a milky white colloidal solution consisting of water (60%), cis-1,4-polyisoprene (35%), and non-isoprene molecules (5%) such as proteins, lipids, carbohydrates, and minerals (Bottier \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Specialized laticifer cells in the bark, leaves, and petioles of the rubber tree synthesize this valuable substance. Commercial latex extraction occurs through a process called tapping, which involves making a controlled incision through half the trunk's circumference at a 30\u0026deg; angle. This precise cut exposes latex vessels in the inner bark while avoiding damage to the cambium. Latex exudes from these incised laticifers and flows along the tapping panel, the channel created by this incision, into collection cups (Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While this method is designed to ensure sustainable production without harming the tree, repeated tapping can sometimes lead to physiological stress and health issues in the tree. One of the most significant challenges faced in this context is Tapping Panel Dryness (TPD), a condition where specific sections or entire tapping panel cease to produce latex despite regular tapping (Herlinawati et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnlike occasional dryness caused by improper tapping or temporary stress, TPD syndrome is more persistent due to irreversible cessation of latex flow and bark necrosis (Herlinawati et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This condition substantially reduces a tree's productive lifespan through permanent blockage of latex biosynthesis and tissue death, severely impacting natural rubber production worldwide.\u003c/p\u003e\u003cp\u003eTPD manifests as a complex syndrome rather than a disease with a single identifiable cause. Despite extensive research, its precise etiology remains elusive. While some studies suggest that the severity of TPD is dependent on the rubber tree clone (Tistama et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the heritability of this trait has not been conclusively established by geneticists and is contested by conflicting reports of environmental stress and overexploitation (Herlinawati et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The clustering pattern of TPD-affected trees observed in plantations and gradual spread of symptoms to adjacent trees over time (Abraham et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), raises the possibility of infectious agents associated with the development of the syndrome. Investigations into potential pathogenic agents, including viruses and viroids, have not provided definitive evidence of their involvement in TPD (Ramachandran et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Studies have reported bark abnormalities such as scaling, cracking, and necrotic streaking in association with TPD, yet comprehensive investigations have failed to implicate any fungal, bacterial, viral, or protozoan agents responsible for its onset (Pellegrin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This persistent knowledge gap necessitates alternative investigative approaches.\u003c/p\u003e\u003cp\u003eRecent systematic investigation from the symptomatology, anatomical studies using confocal and scanning electron microscopy, followed by investigations on physiological changes in the TPD affected bark tissues, strongly suggested the possible presence of phytoplasmas (Philip et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Hence, considering the fundamental role of prokaryotic communities in plant health and disease progression, this study was carried out to verify the phytoplasma presence and to determine whether composition of prokaryotic community in bark tissue differs between healthy and TPD-affected samples.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Collection of samples and study site\u003c/h2\u003e\u003cp\u003eBark samples were collected from a Tapping Panel Dryness (TPD)-affected plantation of clone RRII 430 (Fig. Ia) maintained by the Rubber Research Institute of India (RRII), Kottayam, Kerala, India (9.568689 N, 76.574478 E). The plantation had a TPD incidence rate of approximately 20%, and the trees were 15 years old, with no specific manurial treatments applied. Tapping was conducted using the S2d3 system, where the bark was cut spirally through half the circumference (S2) of the trunk once every three days (d3).\u003c/p\u003e\u003cp\u003eExperimental trees exhibiting typical symptoms of TPD (Fig. Ib \u0026amp; Ic), such as complete blockage of latex flow, scaling, and peeling of bark, were selected as diseased samples. Healthy samples as control were collected from trees with intact bark, regular latex flow, and no visible symptoms of dryness (Fig. Id). Bark samples collected from healthy and TPD affected trees were subjected to anatomical and molecular investigation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Anatomical investigation of healthy and TPD-affected bark samples\u003c/h2\u003e\u003cp\u003eTangential longitudinal sections with an approximate thickness of 35 \u0026micro;m were prepared using a sliding microtome from the inner soft bark region of \u003cem\u003eH. brasiliensis\u003c/em\u003e, a tissue rich in sieve tubes and latex vessels. The sections were then used to examine the presence of callose deposition, latex localization, putative phytoplasma detection, and ultrastructural alterations associated with tapping panel dryness (TPD) syndrome using a combination of histochemical stains, light microscopy, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM).\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Latex vessel staining using Oil Red O\u003c/h2\u003e\u003cp\u003eOil red O based staining was used to identify latex-bearing cells in \u003cem\u003eHevea\u003c/em\u003e (Hamzah et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). Tangential longitudinal sections were treated with 60% isopropanol for 5 minutes followed by staining with 0.5% Oil Red O (w/v) (prepared in 100% isopropanol and diluted to 60% before use). Sections were incubated in the stain for 30 minutes, rinsed briefly in 60% isopropanol, and then mounted in 50% glycerol. Observations were made under a bright field light microscope (LEICA DM 2500 LED).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Callose staining with Congo Red\u003c/h2\u003e\u003cp\u003eTo assess callose deposition in sieve tube elements, sections were stained with 0.1% (w/v) Congo Red (Hi Media, India) for 15 minutes in the dark at room temperature (Sivan et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). After two washes with distilled water, stained sections were mounted in distilled water and examined under a bright field light microscope (LEICA DM 2500 LED)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 CLSM based detection of putative phytoplasma in the sieve tubes of bark sample\u003c/h2\u003e\u003cp\u003eSections were fixed in 4% paraformaldehyde (w/v) in phosphate-buffered saline (PBS; pH 7.2) for 30 minutes at room temperature, washed thoroughly with PBS (3\u0026times;5 min), and stained with 1 \u0026micro;g/mL 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) (Hi Media, India) for 20 minutes in the dark (Andrade and Arismendi \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). After washing in PBS, samples were mounted in 50% glycerol and examined using a confocal laser scanning microscope (CLSM; Leica TCS-SP2, Rajiv Gandhi Centre for Biotechnology, Trivandrum, India) equipped with a UV laser (excitation at 358 nm, emission collected at 461 nm). Images were acquired using Leica Application Suite Advanced Fluorescence software (version 2.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4 Scanning electron microscopy (SEM) based ultrastructural detection of sieve tubes\u003c/h2\u003e\u003cp\u003eTangential longitudinal bark sections were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 hours at 4\u0026deg;C, post-fixed in 1% osmium tetroxide for 1 hour, and dehydrated in an ascending ethanol series (30% to 100%) (Philip et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Samples were critical-point dried using a BAL-TEC CPD 030 Critical Point Dryer \u003cb\u003e(\u003c/b\u003eRajiv Gandhi Centre for Biotechnology, Trivandrum, India\u003cb\u003e)\u003c/b\u003e and mounted on aluminium stubs using double-sided carbon tape. Specimens were sputter-coated with gold in a BAL-TEC SCD 005 sputter coater and examined using a JEOL JSM-6390LV scanning electron microscope operated at 10 and 15 kV.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Metagenomic analysis of prokaryotic communities in healthy and TPD-affected bark samples\u003c/h2\u003e\u003cp\u003eTotal genomic DNA were isolated from bark tissues of healthy and TPD-affected trees and used for 16S rRNA gene-based library preparation and high-throughput sequencing to profile the associated microbial communities. For each condition (healthy and TPD-affected), two trees were selected as biological replicates.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 DNA extraction from the bark samples of healthy and TPD affected trees\u003c/h2\u003e\u003cp\u003eBark samples from multiple portions of the trunk were processed by first removing the outer cortex and grinding the remaining tissue in liquid nitrogen to obtain a fine powder. DNA extraction was performed using a slightly modified version of the protocol described by (Doyle and Doyle \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Approximately 2 g of ground tissue was mixed with preheated CTAB buffer (2% CTAB, 1% polyvinyl pyrrolidone, 1.4 M sodium chloride, 50 mM EDTA, 100 mM Tris-HCl at pH 7.8, and 0.2% β-mercaptoethanol). The lysate was incubated at 65\u0026deg;C for 30 minutes, followed by the addition of an equal volume of chloroform:isoamyl alcohol (24:1) and centrifugation at 15,000g for 25 seconds. The supernatant was carefully collected, treated with 4 \u0026micro;l of RNase, and incubated at 37\u0026deg;C for 30 minutes. DNA was precipitated by adding two-third volume of ice-cold isopropanol. The mixture was centrifuged at 15,000g for 25 seconds to recover the pellet. The resulting pellet was washed with 70% ethanol, air-dried, and dissolved in Milli-Q water. The quality and quantity of the extracted DNA were assessed using agarose gel electrophoresis and Invitrogen Qubit 4 Flurometer for suitability in PCR-based amplification of the 16S rRNA gene. Metagenome sequencing, library preparation and bioinformatic analysis were done with the help of commercial sequencing service providers Eurofins Genomics India, Bengaluru and CloneGtyping, Bengaluru, both from India.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Preparation of 2 x 300 MiSeq library\u003c/h2\u003e\u003cp\u003eThe amplicon library was prepared using the Nextera XT Index Kit. Primers targeting the bacterial-specific region were designed by Eurofins Genomics Lab, India (forward primer: 5\u0026prime; GCCTACGGGNGGCWGCAG 3\u0026prime; and reverse primer: 5\u0026prime; ACTACHVGGGTATCTAATCC 3\u0026prime;). Quality-checked (QC) amplicons with Illumina adapters were further amplified using i5 and i7 primers (Illumina \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which incorporate multiplexing index sequences and common adapters (P5 and P7) (Illumina \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) essential for cluster generation. The amplicon library was purified using AMPure XP beads and quantified with a Qubit Fluorometer. Amplified libraries underwent fragment analysis on the 4200 TapeStation system (Agilent Technologies) using D1000 ScreenTape, following the manufacturer's protocol. Libraries passing QC were sequenced on the MiSeq platform. The resulting sequences from this study have been deposited in the Sequence Read Archive (SRA) under BioProject ID: PRJNA1203434.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Sequence processing, statistical analysis and functional prediction\u003c/h2\u003e\u003cp\u003eThe raw data quality was assessed using the FastQC tool (S. Andrews \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The reads were imported into the QIIME2 environment (Bolyen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) \u003cem\u003evia\u003c/em\u003e the qiime tools import function. After importing, the reads were merged to form a consensus sequence, and a Phred score of 20 was applied as the quality filtering parameter for downstream analyses. Subsequent quality control steps included denoising and dereplication using the qiime vsearch dereplicate-sequences function (Rognes et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which collapsed identical sequences into unique representative sequences. Chimeric sequences were identified and removed using the qiime vsearch uchime-denovo function (Edgar et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Operational Taxonomic Units (OTUs) were clustered de novo at 97% sequence similarity using the qiime vsearch cluster-features-de-novo command, grouping similar sequences into OTUs that represent distinct bacterial taxa. The OTUs were classified using the SILVA 138.1 database. The qiime feature-table summarize function was then used to generate the feature/OTU table.\u003c/p\u003e\u003cp\u003eFor further analysis, the Microeco package (Liu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/ChiLiubio/microeco\u003c/span\u003e\u003cspan address=\"https://github.com/ChiLiubio/microeco\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized. The relative abundance of the top 10 phyla and 12 genera was calculated and visualized using boxplots. Alpha diversity metrics, including observed species, Chao1, ACE, Shannon, Simpson, and Inverse Simpson indices, were computed, with significant variations determined by a t-test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eTo predict the functional potential of the microbial communities, PICRUSt2 (Douglas et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) was employed. Representative sequences in FASTA format and the OTU table (BIOME/TSV format) were exported from QIIME2 for input. The pipeline began by placing Amplicon Sequence Variants (ASVs) onto a reference phylogeny using Hidden-State Prediction (HSP). Gene family abundances, including KEGG Orthologs (KO) and EC numbers, were predicted based on the phylogenetic positioning of the ASVs, and pathway-level predictions were made using MinPath. PICRUSt2 further normalized these predictions to account for variations in 16S rRNA gene copy numbers. The predicted gene families and metabolic pathways were analyzed and visualized using STAMP (Parks et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), providing insights into the functional potential of the microbial communities.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Anatomical differences in the healthy and TPD-affected bark samples of \u003cem\u003eH. brasiliensis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo understand the anatomical changes associated with Tapping Panel Dryness (TPD) in \u003cem\u003eH. brasiliensis\u003c/em\u003e, comparative observations were made between healthy and TPD-affected bark tissues, particularly focusing on sieve tubes, latex vessels, and the presence of intracellular phytoplasma-like bodies. The following subsections detail the findings from each method.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 Visualization of latex vessel organization in healthy and TPD-affected bark samples\u003c/h2\u003e\u003cp\u003eIn healthy bark sections, laticiferous tissues appeared as well-defined, regularly aligned structures, with uniform red staining (Fig. II a). The Oil Red O selectively stained lipid-rich latex vesicles, which were densely packed within the laticifers, indicating normal rubber biosynthesis and storage activity. In contrast, bark tissues from TPD-affected trees (Fig. II b) exhibited noticeable reduction in Oil Red O staining intensity with deformed latex vessel distribution. Dark greyish discoloration was observed in ray parenchyma cells and latex vessels indicating impaired rubber biosynthetic activity or altered metabolic processes associated with the onset of TPD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 Callose deposition in sieve tubes of healthy and TPD-affected bark samples\u003c/h2\u003e\u003cp\u003eSections stained with Congo red were observed for callose deposition. Marked differences were observed between healthy and TPD-affected samples stained with Congo red under bright-field light microscopy. At lower magnification (10X), prominent red staining was localized in the sieve plates of TPD-affected sample (Fig. III). At higher magnification (40X), discrete red bands were observed occluding sieve pores, indicating dense callose deposits across multiple sieve elements (Fig. IV). These occlusions were largely absent or minimal in healthy samples, where sieve plates appeared clear and structurally intact (Fig. IV), indicating free axial and radial transport in the sieve tube.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3 DAPI fluorescence microscopy reveals nuclear material in sieve tubes of TPD-affected sample\u003c/h2\u003e\u003cp\u003eBark sections stained with DAPI, were examined to detect the presence of nuclear material in sieve elements. In healthy bark tissues, no fluorescence signal was observed, indicating the absence of extranuclear DNA in sieve tube elements (Fig. V). In contrast, sections from TPD-affected samples showed prominent DAPI fluorescence localized within sieve elements. These bright, punctate signals suggest the presence of DNA-containing bodies, or microbes potentially corresponding to phytoplasma.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.1.4 Scanning Electron Microscopy (SEM) of sieve tube of TPD-affected bark sample\u003c/h2\u003e\u003cp\u003eSEM imaging of TPD-affected bark sections, at 10 and 15 kV revealed the presence of pleomorphic bodies distributed in the sieve tube at a size ranging from 372 to 991 nm (Fig. VI).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Composition, diversity and functional potential of prokaryotic communities in healthy and TPD-affected samples\u003c/h2\u003e\u003cp\u003eMetagenomic analysis of bark tissues from healthy and TPD-affected \u003cem\u003eH. brasiliensis\u003c/em\u003e trees provided insights into the prokaryotic community structure, diversity, and predicted functional potential, with results described in the following subsections.\u003c/p\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Quantitative and qualitative analysis of DNA and amplicon libraries from healthy and TPD-affected bark samples\u003c/h2\u003e\u003cp\u003eThe DNA yield varied across samples, with healthy and TPD affected bark yielded an average of 128.1 and 59.8 ng/\u0026micro;L, respectively. The purity of all DNA samples was within an acceptable range (260/280 ratio\u0026thinsp;~\u0026thinsp;1.8), ensuring their suitability for amplicon library preparation. Amplification of 16S rRNA generated high quality amplicons of an average size 595 bp which was standardized to 19 ng/\u0026micro;L for sequencing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 Sequencing overview and taxonomic profiling of healthy and TPD-affected bark microbiomes\u003c/h2\u003e\u003cp\u003eSequencing of amplicon libraries generated a substantial number of raw reads, which were subsequently quality filtered to obtain high-confidence sequences of ~\u0026thinsp;301 bp, for taxonomic analysis. In healthy samples, a total of 1,52,547 quality (HQ) reads yielded 2,111 operational taxonomic units (OTUs). The classification of these sequences identified 30 phyla, 93 class, 174 order, 310 family and 500 genera. In contrast, TPD-affected samples exhibited a total of 1,69,807 reads with 2,224 OTUs identified. Their classification revealed a larger microbial diversity in TPD-affected samples, comprising 30 phyla, 92 class, 175 order, 313 family and 505 genera.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 Comparative analysis of bacterial communities in healthy and TPD affected samples\u003c/h2\u003e\u003cp\u003eMicrobial community composition showed no significant differences between healthy and TPD-affected bark samples at any taxonomic level (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Bacteria and Archaea were present in similar proportions, with Proteobacteria and Actinobacteriota as dominant phyla in both conditions. Differences in relative abundances across the major taxa were not statistically significant (Fig. VII, S1\u0026amp;S2). However, a few prokaryotes namely, \u003cem\u003eComamonas, Niastella\u003c/em\u003e, Phytoplasma, \u003cem\u003eSalinibacter\u003c/em\u003e, and \u003cem\u003eWolbachia\u003c/em\u003e were detected exclusively in TPD-affected samples, albeit at very low abundances (\u0026lt;\u0026thinsp;0.01%) (Table. I).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4 Diversity analysis and richness estimates\u003c/h2\u003e\u003cp\u003eAlpha diversity of the samples was analyzed using various diversity indices like Simpson, Chao, Shannon-Wiener, Abundance-based Coverage Estimator (ACE) index, Inverse simpson (Fig. VIII). Species richness was analyzed using Simpson diversity index and Shannon index. Richness of rare species was estimated by Chao 1 and ACE index. Diseased samples exhibited higher variation across all indices as indicated by larger spread in box plots. Consistently lower indices in healthy samples suggest uniformity of microbial community. However, statistically these differences between healthy and infected samples resulted not significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.2.5 Predicted pathways from 16S rRNA\u003c/h2\u003e\u003cp\u003eA total of 7418 PICRUSt2 predicted KEGG orthologs (enzymes) were collapsed to 426 KEGG pathways. The analysis revealed significant alterations in various metabolic pathways between healthy and infected individuals (Fig. IX). In a broader way of categorization in healthy samples, pathways involving amino acid biosynthesis, glycan biosynthesis and metabolism and carbohydrate metabolism resulted highly enriched. Among them, superpathway of methylglyoxal degradation (glycan biosynthesis) and Entner-Doudoroff pathway III (carbohydrate metabolism) were significantly abundant (q\u0026thinsp;\u0026lt;\u0026thinsp;1\u003csup\u003ee\u0026thinsp;\u0026minus;\u0026thinsp;15\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eAmong the top 25 differentially abundant pathways, the superpathway of bacteriochlorophyll a biosynthesis and isopropanol biosynthesis exhibited the most pronounced increase in the infected group, with a significant difference between each other (q\u0026thinsp;\u0026lt;\u0026thinsp;1\u003csup\u003ee\u0026thinsp;\u0026minus;\u0026thinsp;15\u003c/sup\u003e). Additionally, pathways related to formaldehyde assimilation (serine pathway), chlorosalicylate degradation and protocatechuate degradation also showed significant overrepresentation in infected individuals. This suggests enhanced microbial biodegradation of xenobiotics or host metabolites.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study combined anatomical, cytological, metagenomic, and molecular approaches to investigate potential biotic associations with TPD, with a specific focus on vascular tissue integrity and microbial community dynamics. The multidisciplinary findings of the study propose a unified model of TPD pathogenesis and highlights the significance of phytoplasma association in TPD-affected trees.\u003c/p\u003e\u003cp\u003eLatex flow in \u003cem\u003eH. brasiliensis\u003c/em\u003e is highly dependent on the structural and functional integrity of laticifers and their associated phloem tissues (De Fa\u0026yuml; \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Latex vessels stained with Oil red O in the healthy samples appeared structurally intact. As Oil red O has strong affinity for neutral lipids, uniform and intense red staining in healthy samples, indicate abundant and well-preserved lipids in the laticifers (Hamzah et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1988\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCongo red staining of tangential bark sections from healthy \u003cem\u003eH. brasiliensis\u003c/em\u003e revealed well-organized sieve tube elements with clearly visible perforated sieve plates. The sieve plates, which form the interface between adjacent sieve elements, appeared open and unobstructed, allowing unimpeded transport of photosynthates and signaling molecules. Latex vessel absorbs nutrients from neighboring sieve tubes for latex biosynthesis (Sivan et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The absence of callose or phenolic deposition in healthy sample suggests that the phloem is physiologically active and free of stress-induced blockages, maintaining uninterrupted nutrient supply to neighboring tissues, including latex vessels (De Fa\u0026yuml; \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHence, disruption in sieve tube function directly impacts the metabolic activity of adjacent laticifers (Sivan et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Congo Red, a diazo dye with specific affinity for β-1,3-glucans (Piccinini et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), revealed prominent callose deposition and blockage in the sieve plates of TPD-affected bark. Such blockages impair both radial and axial transport in the phloem, leading to nutrient deprivation in latex vessels. This, in turn, disrupts rubber biosynthesis and promotes metabolic stress. Anatomical symptoms such as reduced lipid staining, deformation of latex vessels, and darkened cell contents in TPD-affected samples are consistent with previous studies linking nutrient starvation and oxidative damage in latex vessels due to sieve tube dysfunction (Sivan et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Stress in sieve tubes, whether from biotic or abiotic origins, can thus severely impact latex production. DAPI staining in confocal laser scanning microscopy (CLSM) revealed fluorescence exclusively in TPD-affected samples. As sieve tubes are anucleate and DAPI binds to AT-rich DNA, this fluorescence strongly suggests the presence of microbial DNA, most plausibly phytoplasma, known to inhabit sieve tubes and possess AT-rich genomes (Andrade and Arismendi \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). SEM further confirmed the presence of pleomorphic bodies (319\u0026ndash;990 nm) resembling phytoplasmas in TPD-affected samples. These findings align with previous report of association of phytoplasma with TPD-affected trees of \u003cem\u003eH. brasiliensis\u003c/em\u003e clone RRII 105 (Philip et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e16S rRNA based metagenomic analysis was done to understand the overall prokaryotic composition and diversity in healthy and TPD-affected sample. The bacterial community composition remains consistent across healthy and TPD-affected samples, with no significant shifts in dominant microbial groups at higher taxonomic levels. At the kingdom level, both healthy and TPD-affected samples exhibited similar proportions of Bacteria and Archaea. Various other plants like rice, maize and Scots pine also have reported to be associated with several groups of Archaea in their phytobiome (Jung et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Proteobacteria and Actinobacteria were the most abundant bacterial phyla in both samples. These major phyla are commonly present in almost all ecosystems. Acidobacteria, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes are dominant phyla reported from bark of trees like avocado and red oak (Aguirre-von-Wobeser \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hudson et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe microbial community composition at all taxa remains largely similar in healthy and TPD-affected samples. This suggests that the overall microbiome composition may not be heavily impacted by the disease, or that the disease is not directly associated with large-scale microbial shifts detectable through 16S rRNA amplicon sequencing. Amplicon sequencing, which targets conserved regions (\u003cem\u003ee.g\u003c/em\u003e., 16S rRNA), may not be sensitive enough to detect functional differences or low-abundance microbes that could play a critical role in disease development (Rizal et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOf particular interest is the presence of phytoplasma, with a very low concentration exclusively detected in TPD affected samples. Although detected at extremely low concentrations, phytoplasmas possess the potential to alter host plant metabolism profoundly. Previous studies have shown that concentrations as low as 370 to 34,000 cells per g. of tissue is associated with symptoms of witches\u0026rsquo; broom in apple trees, with nested PCR detecting as few as 4 to 340 cells in reaction mixtures (Berges et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Diseases like, elm yellows, peach X disease, apple proliferation, arecanut lethal yellowing and grapevine bois noir are some of the examples of disease associated with phytoplasma presence in perennial crops with serious economic impact (Wei et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, the minimal concentration of phytoplasmas might explain their low abundance in metagenomic sequencing data, while a possible role in pathogenesis emphasizes their ability to induce disease without significantly altering the microbial diversity or composition in the host microbiome. Recent studies indicate that not all infections lead to major changes in microbial composition. In some cases, like strawberry with signs of plant stress or pathogens, the microbiome showed limited variation between healthy and diseased plants, with only a few taxa displaying noticeable shifts. This suggests that even under pathogenic stress, a stable core microbiome may be retained (Hassani et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhytoplasmas are prokaryotes lacking rigid cell wall. However, their genome size is greatly reduced as a result of reductive evolution from Gram-positive bacteria belonging to Firmicutes. Lack of several genes for basic metabolic pathways and dependence on sucrose as the main source of carbon made them an obligate plant pathogen inhabiting phloem sieve tubes (Oshima \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Given that sieve tubes are rich in sucrose, a precursor for rubber biosynthesis, their blockage due to thick callose and P-protein deposition of TPD-affected trees hint the potential role of phytoplasmas in inducing the syndrome (Sivan et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Buxa-Kleeberg et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Plant defense mechanisms, such as callose deposition and aggregation of P-protein filaments in sieve plates, are reported in plants infected by phytoplasmas (Gallinger and Gross \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nutrient deprivation, coupled with stress-induced defense signaling, could trigger hypersensitive responses and programmed cell death in laticifers, ultimately disrupting latex biosynthesis (Zimmermann et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Meisrimler et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). By linking phytoplasma detection to characteristic symptoms like callose deposition, sieve tube blockage, and laticifer shrinkage, highlights its potential as a critical pathogen underlying TPD, even at low concentrations.\u003c/p\u003e\u003cp\u003eAnalysis revealed that there is no significant variation in microbial diversity indices for healthy and TPD-affected samples. This implies that TPD may not be heavily impacted by the microbial richness and evenness. Amplicon metagenomics often capture overall community composition wherein low titre and localized microbes' effect on richness, evenness and microbial community structure gets unaffected (Cena et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Well supported by the information on symptomatology, pattern of spread, and anatomical changes which are obtained through other experiments in this study, the data on metagenomics suggests or rather detects biologically meaningful trends (Dahlberg et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) indicating the presence of phytoplasmas in TPD.\u003c/p\u003e\u003cp\u003eDespite the similar microbial diversity and composition, there is a significant difference in the metabolic pathways enriched in healthy and TPD affected samples. This could possibly be due to calculation of diversity metrics at a broad taxonomic level (\u003cem\u003ee.g\u003c/em\u003e., genus or species), and they may not be sensitive enough to detect strain-level or functional differences that are important for pathway prediction. However, the metabolic pathways identified in this study were predicted using PICRUSt, which infers functional potential based on more detailed taxonomic resolution or phylogenetic relationships (Sun et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Toole et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). While these results provide valuable insights into potential microbial metabolic shifts, they do not represent direct biochemical activity.\u003c/p\u003e\u003cp\u003ePathways involved in phenolic compound degradation, such as methylgallate degradation, gallate degradation, and chlorosalicylate degradation, were significantly enriched in diseased samples. These compounds are known to play a role in plant defense, and their increased degradation may represent a counter-defense mechanism by pathogens to neutralize plant defenses and establish infections (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Soal et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Phytoplasma infections are known to interfere with host secondary metabolism, particularly phenylpropanoid and salicylic acid pathway\u003cb\u003es\u003c/b\u003e, which could indirectly influence microbial functional dynamics (Pradit et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bauters et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dermastia et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study presents a multidisciplinary investigation into the possible biotic factors associated with TPD in \u003cem\u003eH. brasiliensis\u003c/em\u003e. While there are no major shifts in the dominant microbial groups between healthy and TPD-affected bark, the functional insights from pathway analysis, particularly the enrichment of biodegradative pathways in TPD-affected samples, open avenues for further exploration into the role of microbial metabolism in disease dynamics and possible microbial interactions with host metabolites. Lack of amplification of rare taxa is one of the limitations of amplicon based metagenomic sequencing and hence make it difficult to draw a conclusion on the abundance of microbes. Anatomical and cytological alterations in phloem tissues, alongside metagenomic and PCR based evidences, suggest the presence of a divergent phytoplasma strain in the TPD-affected samples. Although at low concentration, the detection of phytoplasma sequences could be significant, suggesting their potential pathogenic role in TPD. Further studies using targeted and deeper sequencing techniques will elucidate its potential contribution to disease development. Current investigation provides preliminary insights into the biotic complexity of TPD and lay groundwork for future confirmatory studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by Kerala Agricultural University, Thrissur and Rubber research Institute of India, Kottayam.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude to Rubber Research Institute of India for providing laboratory facilities and also acknowledge the valuable assistance of my colleagues Vineeth V. K, Shilpa Babu, Swathi S. J and Rahul Raj for the successful completion of the current research work. The authors are also grateful to Eurofins Genomics India Pvt. Ltd, Bengaluru and CloneGtyping, Bengaluru for metagenome sequencing and bioinformatics analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the authors used free version of Claude in order to improve the writing. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.T. conducted experiment and wrote manuscript text. S.K.P. and S.P. structured the research work and reviewed the manuscript. All other authors reviewed manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbraham A, Philip S, Kuruvilla Jacob C, Jayachandran K (2013) Novel bacterial endophytes from \u003cem\u003eHevea brasiliensis\u003c/em\u003e as biocontrol agent against \u003cem\u003ePhytophthora\u003c/em\u003e leaf fall disease. BioControl 58:675\u0026ndash;684. https://doi.org/10.1007/s10526-013-9516-0\u003c/li\u003e\n\u003cli\u003eAguirre-von-Wobeser E (2020) Functional metagenomics of bark microbial communities from avocado trees (\u003cem\u003ePersea americana\u003c/em\u003e Mill.) reveals potential for bacterial primary productivity. bioRxiv. https://doi.org/10.1101/2020.09.05.284570\u003c/li\u003e\n\u003cli\u003eAlessio FI, Bongiorno VA, Marcone C et al (2025) Genetic Diversity in Phytoplasmas from X-Disease Group Based in Analysis of idpA and imp Genes. Microorganisms 13:1\u0026ndash;14. https://doi.org/10.3390/microorganisms13051170\u003c/li\u003e\n\u003cli\u003eAndrade NM, Arismendi NL (2013) DAPI Staining and Fluorescence Microscopy Techniques for Phytoplasmas. In: Dickinson M, Hodgetts J (eds) Phytoplasma: Methods and Protocols. Humana Press, Totowa, NJ, pp 115\u0026ndash;121\u003c/li\u003e\n\u003cli\u003eBauters L, Stojilković B, Gheysen G (2021) Pathogens pulling the strings: Effectors manipulating salicylic acid and phenylpropanoid biosynthesis in plants. Mol Plant Pathol 22:1436\u0026ndash;1448. https://doi.org/10.1111/mpp.13123\u003c/li\u003e\n\u003cli\u003eBerges R, Rott M, Seemuller E (2000) Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction. Phytopathology 90:1145\u0026ndash;1152. https://doi.org/10.1094/PHYTO.2000.90.10.1145\u003c/li\u003e\n\u003cli\u003eBolyen E, Rideout JR, Dillon MR et al (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37:852\u0026ndash;857. https://doi.org/10.1038/s41587-019-0209-9\u003c/li\u003e\n\u003cli\u003eBottier C (2020) Biochemical composition of \u003cem\u003eHevea brasiliensis \u003c/em\u003elatex: A focus on the protein, lipid, carbohydrate and mineral contents. Adv Bot Res 93:201\u0026ndash;237. https://doi.org/10.1016/bs.abr.2019.11.003\u003c/li\u003e\n\u003cli\u003eBuxa-Kleeberg S, Degola F, Polizzotto R et al (2015) Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements. Front Plant Sci 6:650\u003c/li\u003e\n\u003cli\u003eCena JA de, Zhang J, Deng D et al (2021) Low-Abundant Microorganisms: The Human Microbiome\u0026rsquo;s Dark Matter, a Scoping Review. Front Cell Infect Microbiol 11:. https://doi.org/10.3389/fcimb.2021.689197\u003c/li\u003e\n\u003cli\u003eDahlberg J, Pelve E, Dicksved J (2023) Similarity in milk microbiota in replicates. Microbiologyopen 12:1\u0026ndash;9. https://doi.org/10.1002/mbo3.1383\u003c/li\u003e\n\u003cli\u003eDe Fa\u0026yuml; E (2011) Histo- and cytopathology of trunk phloem necrosis, a form of rubber tree (\u003cem\u003eHevea brasiliensis \u003c/em\u003eMuell. Arg.) tapping panel dryness. Aust J Bot 59:563\u0026ndash;574. https://doi.org/10.1071/BT11070\u003c/li\u003e\n\u003cli\u003eDermastia M, Tomaž \u0026Scaron;, Strah R et al (2023) Candidate pathogenicity factor/effector proteins of \u0026lsquo;\u003cem\u003eCandidatus \u003c/em\u003ePhytoplasma solani\u0026rsquo; modulate plant carbohydrate metabolism, accelerate the ascorbate\u0026ndash;glutathione cycle, and induce autophagosomes. Front Plant Sci 14:1\u0026ndash;16. https://doi.org/10.3389/fpls.2023.1232367\u003c/li\u003e\n\u003cli\u003eDouglas GM, Maffei VJ, Zaneveld JR et al (2020) PICRUSt2 for prediction of metagenome functions. Nat. Biotechnol. 38:685\u0026ndash;688\u003c/li\u003e\n\u003cli\u003eDoyle . J.J., Doyle . J.L. (1987) a Rapid Dna Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochem. Bull. 19:11\u0026ndash;15\u003c/li\u003e\n\u003cli\u003eEdgar RC, Haas BJ, Clemente JC et al (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194\u0026ndash;2200. https://doi.org/10.1093/bioinformatics/btr381\u003c/li\u003e\n\u003cli\u003eFelsenstein J (1985) Confidence Limits on Phylogenies: an Approach Using the Bootstrap. Evolution (N Y) 39:783\u0026ndash;791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x\u003c/li\u003e\n\u003cli\u003eGallinger J, Gross J (2020) Phloem Metabolites of Prunus Sp. Rather than Infection with Candidatus Phytoplasma Prunorum Influence Feeding Behavior of Cacopsylla pruni Nymphs. J Chem Ecol 46:756\u0026ndash;770. https://doi.org/10.1007/s10886-020-01148-8\u003c/li\u003e\n\u003cli\u003eHamzah S, Gomez JB, Ho LH (1988) a Refinement of the Staining Techniques for Hevea Latex Vessels. J Nat Rubber Res 3:163\u0026ndash;166\u003c/li\u003e\n\u003cli\u003eHassani MA, Gonzalez O, Hunter SS, et al (2023) Microbiome Network Connectivity and Composition Linked to Disease Resistance in Strawberry Plants. Phytobiomes J 298\u0026ndash;311. https://doi.org/10.1094/pbiomes-10-22-0069-r\u003c/li\u003e\n\u003cli\u003eHerlinawati E, Montoro P, Ismawanto S et al (2022) Dynamic analysis of Tapping Panel Dryness in \u003cem\u003eHevea brasiliensis \u003c/em\u003ereveals new insights on this physiological syndrome affecting latex production. Heliyon 8:e10920. https://doi.org/10.1016/j.heliyon.2022.e10920\u003c/li\u003e\n\u003cli\u003eHudson JE, Levia DF, Yoshimura KM et al (2023) changes in bark surface phyla. 11:1\u0026ndash;16\u003c/li\u003e\n\u003cli\u003eIllumina (2013) 16S Metagenomic Sequencing Library. Illumina.com 1\u0026ndash;28\u003c/li\u003e\n\u003cli\u003eJung J, Kim JS, Taffner J et al (2020) Archaea, tiny helpers of land plants. Comput Struct Biotechnol J 18:2494\u0026ndash;2500. https://doi.org/10.1016/j.csbj.2020.09.005\u003c/li\u003e\n\u003cli\u003eKawahara S, Nishioka H, Yamano M, Yamamoto Y (2022) Synthetic Rubber with the Tensile Strength of Natural Rubber. ACS Appl Polym Mater 4:2323\u0026ndash;2328. https://doi.org/10.1021/acsapm.1c01508\u003c/li\u003e\n\u003cli\u003eKim M, Oh H-S, Park S-C, Chun J (2014) Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64:346\u0026ndash;351. https://doi.org/10.1099/ijs.0.059774-0\u003c/li\u003e\n\u003cli\u003eKimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111\u0026ndash;120. https://doi.org/10.1007/BF01731581\u003c/li\u003e\n\u003cli\u003eLiu C, Cui Y, Li X, Yao M (2020) microeco : An R package for data mining in microbial community ecology. FEMS Microbiol Ecol 97:. https://doi.org/10.1093/femsec/fiaa255\u003c/li\u003e\n\u003cli\u003eMeisrimler CN, Allan C, Eccersall S, Morris RJ (2021) Interior design: how plant pathogens optimize their living conditions. New Phytol 229:2514\u0026ndash;2524. https://doi.org/10.1111/nph.17024\u003c/li\u003e\n\u003cli\u003eOshima K (2021) Molecular biological study on the survival strategy of phytoplasma. J Gen Plant Pathol 87:. https://doi.org/10.1007/s10327-021-01027-4\u003c/li\u003e\n\u003cli\u003eOshima K, Kakizawa S, Nishigawa H et al (2004) Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nat Genet 36:27\u0026ndash;29. https://doi.org/10.1038/ng1277\u003c/li\u003e\n\u003cli\u003eParks DH, Tyson GW, Hugenholtz P, Beiko RG (2014) STAMP: statistical analysis of taxonomic and functional profiles. Bioinformatics 30:3123\u0026ndash;3124. https://doi.org/10.1093/bioinformatics/btu494\u003c/li\u003e\n\u003cli\u003ePellegrin, F. Nandris, D. and Chrestin H (2004) Rubber Tree (\u003cem\u003eHevea brasiliensis\u003c/em\u003e) Bark Necrosis Syndrome I: Still No Evidence of a Biotic Causal Agent. Plant Dis 88:1,046.3-1,046.3. https://doi.org/http://dx.doi.org/10.1094/PDIS.2004.88.9.1046C\u003c/li\u003e\n\u003cli\u003ePiccinini L, Nirina Ramamonjy F, Ursache R (2024) Imaging plant cell walls using fluorescent stains: The beauty is in the details. J Microsc 295:102\u0026ndash;120. https://doi.org/10.1111/jmi.13289\u003c/li\u003e\n\u003cli\u003ePradit N, Rodriguez-Saona C, Kawash J, Polashock J (2019) Phytoplasma Infection Influences Gene Expression in American Cranberry. Front Ecol Evol 7:1\u0026ndash;14. https://doi.org/10.3389/fevo.2019.00178\u003c/li\u003e\n\u003cli\u003ePuskas JE, Cornish K, Kenzhe-Karim B et al (2024) Natural rubber \u0026ndash; Increasing diversity of an irreplaceable renewable resource. Heliyon 10:e25123. https://doi.org/https://doi.org/10.1016/j.heliyon.2024.e25123\u003c/li\u003e\n\u003cli\u003eRamachandran P, Mathur S, Francis L et al (2000) Evidence for Association of a Viroid with Tapping Panel Dryness Syndrome of Rubber (Hevea brasiliensis). Plant Dis 84:1155. https://doi.org/10.1094/PDIS.2000.84.10.1155C\u003c/li\u003e\n\u003cli\u003eRizal NSM, Neoh HM, Ramli R et al (2020) Advantages and limitations of 16S rRNA next-generation sequencing for pathogen identification in the diagnostic microbiology laboratory: perspectives from a middle-income country. Diagnostics 10:. https://doi.org/10.3390/diagnostics10100816\u003c/li\u003e\n\u003cli\u003eRognes T, Flouri T, Nichols B et al (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. https://doi.org/10.7717/peerj.2584\u003c/li\u003e\n\u003cli\u003eAndrews S (2010) FastQC: a quality control tool for high throughput sequence data\u003c/li\u003e\n\u003cli\u003ePhilip S, Tom A, Prem E, Puramerimadathil R, Sajeed RK (2025) Association of phytoplasmas with tapping panel dryness syndrome of \u003cem\u003eHevea brasiliensis\u003c/em\u003e: a pathological perspective to unresolved mystey? Phytopathogenic Mollicutes 15:63\u0026ndash;64\u003c/li\u003e\n\u003cli\u003eSivan P, Thomas V, Rao K, Krishnakumar R (2011) Definitive Callose Deposition in Tapping Panel Dryness Affected Bark of \u003cem\u003eHevea brasiliensis\u003c/em\u003e. J Sustain For 30:329\u0026ndash;342. https://doi.org/10.1080/10549811.2011.532032\u003c/li\u003e\n\u003cli\u003eSmart CD, Schneider B, Blomquist CL et al (1996) Phytoplasma-Specific PCR Primers Based on Sequences of the 16S-23S rRNA Spacer Region. 62:2988\u0026ndash;2993\u003c/li\u003e\n\u003cli\u003eSoal NC, Coetzee MPA, van der Nest MA et al (2022) Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae. G3 Genes, Genomes, Genet 12:. https://doi.org/10.1093/g3journal/jkac008\u003c/li\u003e\n\u003cli\u003eSun S, Jones RB, Fodor AA (2020) Inference-based accuracy of metagenome prediction tools varies across sample types and functional categories. Microbiome 8:1\u0026ndash;9. https://doi.org/10.1186/s40168-020-00815-y\u003c/li\u003e\n\u003cli\u003eTamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 38:3022\u0026ndash;3027. https://doi.org/10.1093/molbev/msab120\u003c/li\u003e\n\u003cli\u003eThangamalai A (2021) Growth and Prospects of Natural and Synthetic Rubber Production and Consumption in India. Rev Gest\u0026atilde;o Inova\u0026ccedil;\u0026atilde;o e Tecnol 11:2019\u0026ndash;2032. https://doi.org/10.47059/revistageintec.v11i4.2251\u003c/li\u003e\n\u003cli\u003eThompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673\u0026ndash;4680. https://doi.org/10.1093/nar/22.22.4673\u003c/li\u003e\n\u003cli\u003eTistama R, Mawaddah PAS, Ade-Fipriani L, Junaidi (2019) Physiological status of high and low metabolism Hevea clones in the difference stage of tapping panel dryness. Biodiversitas 20:267\u0026ndash;273. https://doi.org/10.13057/biodiv/d200143\u003c/li\u003e\n\u003cli\u003eToole DR, Zhao J, Martens-Habbena W, Strauss SL (2021) Bacterial functional prediction tools detect but underestimate metabolic diversity compared to shotgun metagenomics in southwest Florida soils. Appl Soil Ecol 168:104129. https://doi.org/10.1016/j.apsoil.2021.104129\u003c/li\u003e\n\u003cli\u003eWang L, Huang C, Li T et al (2023) An Optimization Study on a Novel Mechanical Rubber Tree Tapping Mechanism and Technology. Forests 14:1\u0026ndash;25. https://doi.org/10.3390/f14122421\u003c/li\u003e\n\u003cli\u003eWang R, Bai B, Li D et al (2024) Phytoplasma: A plant pathogen that cannot be ignored in agricultural production\u0026mdash;Research progress and outlook. Mol Plant Pathol 25:1\u0026ndash;19. https://doi.org/10.1111/mpp.13437\u003c/li\u003e\n\u003cli\u003eWei W, Inaba J, Zhao Y et al (2022) Phytoplasma Infection Blocks Starch Breakdown and Triggers Chloroplast Degradation, Leading to Premature Leaf Senescence, Sucrose Reallocation, and Spatiotemporal Redistribution of Phytohormones. Int J Mol Sci 23:. https://doi.org/10.3390/ijms23031810\u003c/li\u003e\n\u003cli\u003eZhang J, Coaker G, Zhou JM, Dong X (2020) Plant Immune Mechanisms: From Reductionistic to Holistic Points of View. Mol Plant 13:1358\u0026ndash;1378. https://doi.org/10.1016/j.molp.2020.09.007\u003c/li\u003e\n\u003cli\u003eZhang Q, Li M, Yang G et al (2022) Protocatechuic acid, ferulic acid and relevant defense enzymes correlate closely with walnut resistance to \u003cem\u003eXanthomonas arboricola\u003c/em\u003e pv. \u003cem\u003ejuglandis.\u003c/em\u003e BMC Plant Biol 22:1\u0026ndash;14. https://doi.org/10.1186/s12870-022-03997-9\u003c/li\u003e\n\u003cli\u003eZhang Y, Leclercq J, Montoro P (2016) Reactive oxygen species in \u003cem\u003eHevea brasiliensis\u003c/em\u003e latex and relevance to Tapping Panel Dryness. 2:261\u0026ndash;269. https://doi.org/10.1093/treephys/tpw106\u003c/li\u003e\n\u003cli\u003eZhao R, Su X, Yu F et al (2023) Identification and characterization of two closely related virga-like viruses latently infecting rubber trees (\u003cem\u003eHevea brasiliensis\u003c/em\u003e). Front Microbiol 14:1286369. https://doi.org/10.3389/fmicb.2023.1286369\u003c/li\u003e\n\u003cli\u003eZimmermann MR, Schneider B, Mith\u0026ouml;fer A et al (2015) Implications of \u003cem\u003eCandidatus\u003c/em\u003e Phytoplasma mali infection on phloem function of apple trees. J Endocytobiosis Cell Res 26:67\u0026ndash;75\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Core microbiome, Para rubber, Phytoplasma, Natural rubber, TPD","lastPublishedDoi":"10.21203/rs.3.rs-8276740/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8276740/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTapping Panel Dryness is a complex physiological disorder in \u003cem\u003eHevea brasiliensis\u003c/em\u003e that leads to the cessation of latex flow, causing significant economic loss, yet its underlying cause remains unclear. Anatomical investigation of bark samples collected from TPD-affected samples exhibited deformed latex vessels, blocked sieve tubes, and DNA-containing bodies within phloem elements. Metagenomic profiling indicated largely similar microbial composition and diversity between healthy and TPD-affected bark samples, except for the presence of low-abundance taxa such as phytoplasma only in affected samples. However, predicted metabolic pathways differed significantly between healthy and TPD samples. 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