Histochemical Analysis for Lignin Differences Between Reniform Nematode-Resistant and Susceptible Cotton Genotypes

Summary

Reniform nematode infection significantly reduces cotton yield and fiber quality. Lignin strengthens plant cell walls and contributes to structural defenses against pests, but its role in reniform nematode resistance in cotton has not been examined. This study describes a histochemical workflow for visualizing lignin deposition in roots of reniform nematode-resistant and susceptible cotton (Gossypium arboreum) genotypes using phloroglucinol staining.

Abstract

Reniform nematodes (Rotylenchulus reniformis) are a major constraint on cotton production, particularly in the southern United States. The reniform nematode is the second most important pest among more than 400 nematode species. These nematodes pose a threat to global food security. Increasing environmental and human health concerns and the cost of nematicides have intensified interest in resistant cotton germplasm. The plant cell wall, especially lignin, is a critical biopolymer involved in resistance to biotic stress. Because nematodes infect root tissues, cell wall components such as lignin may contribute to defense; however, lignin-mediated barriers in cotton roots are not well characterized. This study employed a standardized phloroglucinol-HCl histochemical staining protocol to visualize lignin deposition differences in reniform nematode-resistant (A2-514, A2-995) and susceptible cotton (Gossypium arboreum) genotypes at the true leaf stage under non-infected conditions. This stain binds specifically to hydroxy cinnamylaldehyde such as coniferyl aldehyde and sinapyl aldehyde of lignin in the presence of acid and generates a pinkish purple color to visualize lignin. Distinct lignin staining patterns were observed between resistant and susceptible genotypes, supporting a potential role for lignin-associated structural defenses.

Introduction

Cotton is cultivated worldwide for its fiber and is highly vulnerable to soil-borne pests, particularly nematodes, which can cause substantial yield losses. Among these, the reniform nematode (Rotylenchulus reniformis) is a major pathogen affecting cotton and other crops globally, with pronounced impacts in the southern United States, where infestations can reduce yields by more than 50%¹. This microscopic, soil-dwelling organism parasitizes cotton roots², impairing plant vigor, biomass, and fiber yield through disruption of water and nutrient uptake and the establishment of feeding sites (Figure 1). Reniform nematode infection alters cotton plant growth by delaying flowering time, reducing boll size and number, and compromising lint quality³. Severe losses have been reported in Mississippi, Alabama, and Louisiana⁴. Nematicides are commonly employed to manage nematode infestations, but their high cost and negative impact on the environment and human health have necessitated the need for alternatives^5,6. The development of resistant germplasm represents a promising approach to mitigate reniform nematode damage⁴. Because nematodes feed within root tissues, cell-wall-based defenses, particularly lignification, are hypothesized to restrict invasion, inhibit pathogen-derived cell-wall-degrading enzymes, and limit the establishment and expansion of nematode feeding sites^7,8,9. Enhanced lignification has been correlated with reduced pathogen susceptibility across plant species^{10,11,12,13,14}. Prior research in cotton has linked lignin and laccase gene expression to resistance against Verticillium dahliae¹⁵ and studies in Fusarium resistant and susceptible cultivars revealed higher lignin levels in resistant cultivars¹⁶. Similar observations in Arabidopsis and rice, where suppression of lignin biosynthetic genes increased susceptibility, underscore lignification’s importance in plant defense.

Lignin is a complex phenolic biopolymer found in the secondary cell walls of vascular plants, where it imparts structural rigidity, mechanical strength, and hydrophobicity¹⁷. Synthesized through the shikimate and phenylpropanoid pathways, lignin is the second most abundant terrestrial biopolymer after cellulose, accounting for approximately 30% of the earth’s organic carbon^18,19. By reinforcing the cellulose-hemicellulose network, lignin strengthens load-bearing tissues and supports overall plant development. Lignin is especially prominent in highly specialized tissues such as xylem, sclerenchyma, phloem fibers, and periderm, creating a water‑repellent barrier essential for efficient transport of water and minerals. In addition to structural functions, lignin acts as a biochemical and physical defense barrier, restricting pathogen ingress and minimizing damage from biotic and abiotic stresses^20,21,22. Lignification is a dynamic process central to resistance against a wide spectrum of pathogens, including necrotrophs, biotrophs, and hemibiotrophs. Its biosynthesis from the primary monolignols sinapyl, coniferyl, and p-coumaryl alcohol varies by species, tissue type, and developmental stage, influencing the degree of resistance. Pathogen invasion or abiotic stress often triggers localized lignin deposition, further fortifying plant defenses^23,24.

Elucidating lignin’s role in reniform nematode resistance is crucial for advancing cotton management strategies. Visual and biochemical characterization of lignin distribution offers insight into the spatial and temporal dynamics of cell‑wall modifications that underpin early defense responses. This study employs histochemical phloroglucinol staining to analyze lignin deposition patterns that naturally exist in reniform‑nematode‑resistant and susceptible cotton genotypes, providing a practical preliminary framework for evaluating cell-wall-associated resistance traits and informing breeding programs aimed at improving crop resilience.

Protocol

The experiments were conducted in a greenhouse using the plant materials described below. All chemicals, equipment, and reagents utilized in this study are detailed in the Table of Materials.

Plant material

Seeds were obtained for both susceptible (A2‑20, PI 129742; A2‑40, PI 183202) and resistant germplasm (A2‑514, PI 616008; A2‑995, PI 629323). Additionally, Deltapine 16, a susceptible cultivar, was included in this study. Seeds were placed in sterile water and soaked for 24 h at room temperature. Following soaking, seeds were thoroughly rinsed with fresh sterile water to remove residual contaminants. The rinsed seeds were then transferred onto sterile Petri plates lined with pre‑wetted, autoclaved paper towels and incubated for 2 days at room temperature to facilitate germination (Figure 2).

Greenhouse planting

Plastic pots were filled with pre-moistened moisture-control potting mix. The soil was pre-wetted for two days prior to transplanting. Germinated seedlings were then carefully transplanted into the pre‑wetted soil and maintained under controlled greenhouse conditions at 28 °C/25 °C day/night cycles until the true-leaf stage (Figure 2). Three biological replicates were planted for each genotype.

Sample collection

Plants at the true‑leaf stage were gently removed from the soil by loosening the surrounding soil medium to minimize root damage. Roots were rinsed 2–3 times with water to remove adhering soil particles. Root and shoot tissues from each genotype were collected for sectioning (Figure 3).

Preparation of phloroglucinol staining solution

A 3% phloroglucinol solution was prepared by dissolving 0.45 g of phloroglucinol in a mixture of two parts 100% ethanol (10 mL) and one part concentrated HCl (5 mL) in a fume hood. The staining solution was prepared following a previously described method¹⁶.

Preparation of section and staining procedure for histochemical analysis of lignin

Root and shoot tissues were sectioned using a sterile razor blade to obtain thin transverse sections (Figure 3). Fresh sections were transferred to microscope slides and covered with 20 µL of the 3% phloroglucinol staining solution for 1 min. Sections were then rinsed with sterile water, mounted under coverslips, and imaged (Figure 4, Figure 5, Figure 6). The same procedure was used for stem tissues (Supplementary Figure 1).

Microscopy imaging

Imaging was performed using a digital microscope. Prepared glass slides containing stained tissue samples were placed on the microscope platform for imaging. Following initialization of the microscope imaging software, the desired magnifications of 100x, 200x, and 300x were selected in bright-field mode. Coarse and fine focus adjustments were made to clearly visualize lignin staining patterns within the tissue sections. Images were recorded and saved with their respective sample identification numbers. Comparative analysis was then performed by examining images from both resistant and susceptible genotypes to assess differences in lignin staining. Images of resistant and susceptible genotypes were compared using ImageJ to determine differences in the percent stained area using uniform thresholds. Results were exported as CSV files after all three replicates were processed. Graphs were then prepared to compare resistant and susceptible genotypes.

Results

Histochemical analysis of resistant and susceptible cotton genotypes

Histochemical examination of transverse root and stem sections at true leaf stage revealed visual differences in resistant and susceptible genotypes as demonstrated in Figure 4, Figure 5, and Supplementary Figure 1 (stem transverse sections). Higher magnifications (100x–300x) provided detailed views of these differences (Figure 4A–C and Figure 5A–C for resistant genotypes and Figure 4D–F and Figure 5D–F for susceptible genotypes). Lignin staining patterns differed by tissue types: resistant genotypes exhibited lower or similar lignin staining patterns in stems, while susceptible genotypes displayed relatively higher stem lignification visually (Supplementary Figure 1A–D).

Quantitative image analysis of lignin staining

Deltapine 16, a susceptible cultivar included in this study, displayed lower lignin staining (Figure 6A–C) with percent stained area values of 12, 17.8, and 16.3 (Figure 6D). The susceptible genotype A2-40 had percent stained area values of 25.2, 19.9, and 28.75, while the resistant genotype, A2-995, showed percent stained area values of 54.95, 58.71, and 69.85 (Figure 7). However, A2-20 had percent stained area values of 16.6, 21.3, and 20.4 whereas A2-514 showed percent stained area values as 49.8, 34.1, and 43.38 (Figure 8). The comparison yielded a p-value of 0.06. Because these values were generated using different thresholds, uniform-threshold ImageJ analysis was performed using identical hue, saturation, and brightness thresholds to ensure reproducibility across replicates. The resistant genotype (A2-514) showed relatively greater staining than the susceptible genotype (A2-20). Percent stained area was calculated as the ratio of lignin-positive pixels to total tissue pixels within the section. The uniform-threshold quantitative comparison of the susceptible genotype A2-20 and resistant genotype A2-514 is shown in Figure 9. Overall, uniform threshold analysis demonstrates that genotype-specific lignification differences exist among these genotypes.

DATA AVAILABILITY:

Raw data is added as a Supplementary File.

Figure 1: Microscopic view of reniform nematodes feeding on cotton roots. Reniform nematodes feeding on cotton roots are shown at 50x magnification following staining with red food coloring. Please click here to view a larger version of this figure.

Figure 2: Preparation of plant material for lignin histochemical analysis. The workflow consists of the following steps: (1) soak cotton seeds in water; (2) place the seeds on wet paper towels in a labeled sterile Petri plate; (3) allow the seeds to germinate for 2 days and transfer the seedlings to labeled pots containing soil; (4) grow the seedlings to the cotyledon stage; (5) continue growing the plants to the true-leaf stage and prepare cross sections of the stems and roots; and (6) stain the collected tissue sections and observe them under a microscope. Please click here to view a larger version of this figure.

Figure 3: Collection and staining of cotton root and stem sections using phloroglucinol-HCl. The workflow consists of the following steps: (1) grow three biological replicates to the true-leaf stage; (2) gently remove the plants from the soil, wash with water, and prepare them for sectioning; (3) use a sterile blade to prepare transverse stem sections; (4) use a sterile blade to prepare transverse root sections; (5) stain the stem sections with 3% phloroglucinol-HCl, rinse with water, and observe under a microscope; (6) stain the root sections with 3% phloroglucinol-HCl, rinse with water, and observe under a microscope; and (7) observe the stained sections under a microscope to visualize phloroglucinol staining differences and record images. Please click here to view a larger version of this figure.

Figure 4: Lignin staining in transverse root sections of A2-514 and A2-20 cotton genotypes. Lignin-stained transverse root sections of the resistant genotype A2-514 at (A) 100x, (B) 200x, and (C) 300x magnification. Lignin-stained transverse root sections of the susceptible genotype A2–20 at (D) 100x, (E) 200x, and (F) 300x magnification. Please click here to view a larger version of this figure.

Figure 5: Lignin staining in transverse root sections of A2-995 and A2-40 cotton genotypes. Lignin-stained transverse root sections of the resistant genotype A2-995 at (A) 100x, (B) 200x, and (C) 300x magnification. Lignin-stained transverse root sections of the susceptible genotype A2-40 at (D) 100x, (E) 200x, and (F) 300x magnification. Please click here to view a larger version of this figure.

Figure 6: Lignin staining and quantitative analysis of transverse root sections of Deltapine 16. Lignin-stained transverse root sections of three biological replicates of the susceptible cultivar Deltapine 16 at 100x magnification: (A) biological replicate 1, (B) biological replicate 2, and (C) biological replicate 3. (D) Percent stained area for the three biological replicates determined by quantitative ImageJ analysis. Please click here to view a larger version of this figure.

Figure 7: Quantitative analysis of lignin staining in A2-40 and A2-995 cotton genotypes. Percent stained area determined by ImageJ analysis for three biological replicates of the susceptible genotype A2-40 and the resistant genotype A2-995. Please click here to view a larger version of this figure.

Figure 8: Quantitative analysis of lignin staining in A2-20 and A2-514 cotton genotypes. Percent stained area determined by ImageJ analysis for three biological replicates of the susceptible genotype A2-20 and the resistant genotype A2-514. Please click here to view a larger version of this figure.

Figure 9: Uniform-threshold quantification of lignin staining in A2-20 and A2-514 cotton genotypes. Percent stained area in the susceptible genotype A2-20 and resistant genotype A2-514 following uniform-threshold image analysis. Statistical comparison was performed using Welch's t-test (t = −1.52, p = 0.267). Please click here to view a larger version of this figure.

Figure 10: Visualization of lignin staining across different root regions. Representative images showing lignin staining in the root maturation, root elongation, and root tip regions. Please click here to view a larger version of this figure.

Supplementary Figure 1: Lignin staining in transverse stem sections of resistant and susceptible cotton genotypes. Lignin-stained transverse stem sections at 100x magnification of (A) susceptible A2-20, (B) resistant A2-514, (C) susceptible A2-40, and (D) resistant A2-995. Please click here to download this file.

Discussion

Lignin associated with plant cell walls serves as an important physical barrier against pests, including nematodes. In the present study, resistant and susceptible cotton genotypes showed distinct visual differences in lignin staining, following a pattern similar to that previously reported in Fusarium-resistant and susceptible cultivars. The staining allowed clear visualization of lignin distribution across root regions—maturation, elongation, and the root tip—where lignin intensity increased progressively from the upper root toward the tip (Figure 10). While these patterns provide useful anatomical insight, lignin dynamics in response to nematode infection were not evaluated in the current study and require confirmation in future research. Previous studies suggest that pathogen presence can induce lignin biosynthesis, reinforcing the need to examine lignin changes under nematode pressure. Incorporating biochemical analyses of lignin quantity and composition would further strengthen interpretation of the staining patterns observed in these genotypes. Overall, these findings highlight the role of lignin-mediated structural defenses in cotton reniform nematode resistance and demonstrate the utility of phloroglucinol staining for characterizing these traits. Continued integration of anatomical, biochemical, and molecular approaches will deepen the understanding of lignin biosynthesis and its contribution to developing durable resistance to reniform nematodes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Moisture control potting mix	Miracle-Gro	073561005197 032247557832	Amazon web site
Microscope	Keyence VHX	VHX3000	Any comparable bright field microscope can be used
Sterile Razor blades	Workpro	W018057	Amazon web site
Cutting board	Freshware	15.75"L x 9.8"W x 0.3"Th	Amazon web site
Slides and coverslips	Amscope	BS-50P-100S-22X4	Any brand can be used. This one is available at amazon.
Sterile water/ Distilled water/ autoclaved water	Gibco	15230147	Any brand/ sterile atoclaved water can be used
Phloroglucinol stain	Fischer Scientific	AC241761000	Available at Fischer Scientific
Concentrated HCl	Fischer Scientific	CAS: 7647-01-0	Available at Fischer Scientific
Absolute ethanol	Fischer Scientific	64-17-5	Available at Fischer Scientific
Pots	BTPLASTIK	B0DSBQMXHN	Any plastic pots can be used, availale at amazon
Petri plates	Kingdder	B0BS95MCGS	Available at amazon and any other brand can be used
Paper towels	Tork	MB540A	Available at amazon and any other brand can be used
Image J	Software	Available online	Free software available to use