Species variability of Pinus elliottii, P. elliottii×P. caribaea, and P. massoniana in response to pinewood nematode infection

Plant material

The study was conducted in a nursery located in Tianhe District, Guangzhou City (113°37′E, 23°18′N). The site is situated at an elevation of 25 m above sea level and experiences a typical subtropical monsoon climate. The average annual temperature is about 22 °C, with January and July being the coldest and hottest month. The region receives an average annual rainfall of approximately 1,800 mm, with the majority occurring during the summer months. The inoculations were carried out in greenhouse conditions (average temperature of 26° ±2 °C, 60–80% humidity) at the nursery.

This study involved one-year-old seedlings (100 individuals of each species) of P. elliottii (72.5 ± 0.6 cm height, 8.05 ± 0.12 mm ground diameter), P. elliottii × P. caribaea hybrids (87.8 ± 1.1 cm height, 9.60 ± 0.21 mm ground diameter), and P. massoniana (50.3 ± 1.1 cm height, 9.60 ± 0.21 mm ground diameter). The pine seedlings used in this study were nursery-grown from seeds collected from seed orchards of the three tested species in Guangdong Province. Once germinated and established, the seedlings were transplanted to an isolated nursery for further growth. Two weeks before inoculation, the seedlings were moved into the greenhouse. The plants were watered twice every day, ensuring that the soil remained adequately moist but avoiding waterlogging. Watering was conducted at a consistent rate across all treatment groups to maintain uniform soil moisture conditions.

Culture of pinewood nematode

The B. xylophilus isolate was a virulent PWN cultured by the South China Agricultural University. The nematodes were maintained and propagated on a fungal culture of Botrytis cinerea, grown on potato dextrose agar (PDA) in Petri dishes under laboratory conditions.

To establish nematode cultures, actively feeding nematodes were transferred to the fungal colonies using a sterilized fine needle. The plates were incubated at 25 ± 1 °C for 7–10 days, during which the nematodes proliferated. After sufficient nematode growth, the nematodes were extracted using the Baermann funnel technique. Extracted nematodes were rinsed with sterile distilled water, quantified under a stereomicroscope, and used for subsequent inoculations.

Inoculation method and experimental setup

We used a serrated-incision inoculation method adapted from previous studies (Togashi et al., 1997; Carrasquinho et al., 2018; Menéndez-Gutiérrez et al., 2018b), making a cut 20 cm above the ground to mimic Monochamus-beetle wounding and facilitate nematode entry (Fig. 1A). Unlike the original protocols, our streamlined approach omits wound-moisture treatments and reduces handling time while still ensuring consistent nematode delivery and effective migration into the plant. The nematode suspension (100 µL) was applied directly to the exposed xylem. Three inoculation concentrations were used: 2,000 nematodes per tree, 4,000 nematodes per tree, and 8,000 nematodes per tree. A control group was treated with distilled water. Each treatment for each tree species included 24 replicates.

Disease observation and severity index

After inoculation, the disease symptoms were recorded weekly, and each tree was assigned a disease severity index (0 to 4), where 0 indicated no symptoms, and 4 indicated severe wilting and chlorosis of more than 75% of the tree. The susceptibility rate (Rempel & Hall, 1996; Qiu et al., 2023) and mortality rate were calculated for each treatment group using the following formulas: ${\displaystyle \text{Susceptibility rate}=\left(\text{number of symptomatic trees}/\text{total number of trees}\right)\times 100\text{\%}}$ ${\displaystyle \text{Mortality rate}=\left(\text{number of dead trees}/\text{total number of trees}\right)\times 100\text{\%}.}$

Stem section observation and nematode migration analysis

Based on the results of preliminary experiments, B. xylophilus infection was found to cause structural damage throughout the entire stem of the seedlings. To ensure consistency in sampling and facilitate comparative analysis across different treatments, the position five cm above the base of the stem was selected for cross-sectional microscopy. Stem samples were collected at 35 days post inoculation from the stem of each plant, precisely five cm above the base. Cross-sectional observations were conducted using a DSX100 ultra-depth three-dimensional microscope (Olympus, Tokyo, Japan) to assess anatomical differences in the xylem and associated tissues. Structural variations between xylem tissues in plants with four grades of disease severity and those from the control group were systematically compared. Observations focused on the disruption of xylem integrity, resin canals, and other tissue alterations caused by nematode infection.

To evaluate nematode migration and reproduction within the plant, wood segments were collected above and below the inoculation site for the symptomatic trees also at 35 days post inoculation. A 10-cm segment was excised in both directions after removing the wound area. The fresh weight of each segment was measured, and the samples were finely chipped. Nematodes were extracted from the wood chips using the Baermann funnel method, with the setup maintained for 24 h to allow nematode migration into the extraction solution. The nematode-containing solution was collected and centrifuged at 5,000 rpm for 10 min. After removing the supernatant, the nematode pellet was resuspended in two mL of distilled water to prepare the identification solution. Three 50 µL subsamples from the identification solution were prepared on microscope slides for nematode counting under a stereomicroscope. Three biological replicates were included for each treatment, and counts were averaged across the subsamples. The density of nematodes within each wood segment was calculated using the formula: ${\displaystyle \text{Nematode Density}\left(/g\text{wood}\right)=\text{Nematode count on slides}\times \left(\text{Total identification solution volume}/50\right)/\text{Fresh weight of wood}.}$

Statistical analysis

Statistical analyses were performed using the Statistical Product and Service Solutions (SPSS) 21.0 software. Data are expressed as the mean ± standard error (SE). Variance among multiple groups was compared using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post-test. Statistical significance was considered at P < 0.05. Graphical representations of the data were generated using Microsoft Excel or OriginPro 8.0.

Symptom development across tree species

Following inoculation with B. xylophilus, initial symptoms began to appear approximately one week post-inoculation, progressively intensifying over time. The severity of symptoms varied among species, with clear differences in disease progression and symptom expression (Figs. 1B–1D). Throughout the observation period, individuals within each species exhibited varying degrees of susceptibility, as indicated by the presence of trees across all disease severity index (DSI) levels.

At DSI 0, all species maintained healthy needle color and structure, showing no visible symptoms of infection (Figs. 1B–1D, first column). By DSI 1, P. massoniana exhibited mild chlorosis and noticeable needle drooping, particularly on lower branches (Fig. 1B, second column). In contrast, P. elliottii × P. caribaea showed yellowing predominantly in the upper canopy, with slight drooping in some branches (Fig. 1C, second column). P. elliottii, however, mainly exhibited needle yellowing without significant drooping, maintaining an upright structure (Fig. 1D, second column).

As infection progressed to DSI 2, symptoms intensified across all species. P. massoniana displayed widespread needle yellowing and increased drooping, particularly in the lower branches (Fig. 1B, third column). P. elliottii × P. caribaea showed a more symmetrical pattern of yellowing, with partial drooping but a relatively intact canopy structure (Fig. 1C, third column). In P. elliottii, needle yellowing became more pronounced, yet the tree maintained its structural integrity, with minimal branch drooping (Fig. 1D, third column).

At DSI 3, P. massoniana exhibited extensive needle browning, severe wilting, and overall plant collapse, with many branches losing their turgidity (Fig. 1B, fourth column). P. elliottii × P. caribaea also showed severe chlorosis and wilting, but some green foliage remained in the upper canopy (Fig. 1C, fourth column). Meanwhile, P. elliottii continued to display dominant yellowing symptoms rather than extensive drooping, differentiating it from the other two species (Fig. 1D, fourth column).

By DSI 4, P. massoniana was the most severely affected, with extreme wilting, extensive chlorosis, and complete needle loss, often leading to plant death (Fig. 1B, fifth column). P. elliottii × P. caribaea exhibited similar mortality symptoms, with its structure collapsing but some branches retaining discolored needles (Fig. 1C, fifth column). In contrast, P. elliottii, while exhibiting full chlorosis and needle desiccation, maintained a more upright structure, suggesting a different mode of symptom expression compared to the other two species (Fig. 1D, fifth column).

These results indicate that while all species were affected by B. xylophilus infection, P. massoniana was the most susceptible, with rapid symptom development and extensive wilting. P. elliottii × P. caribaea showed moderate susceptibility, exhibiting chlorosis and partial drooping, while P. elliottii demonstrated the highest resistance, primarily displaying chlorosis with minimal needle drooping.

Changes in xylem and pith structures

The effects of B. xylophilus infection on the xylem and pith were assessed by examining stem cross-sections from both healthy and infected plants (Fig. 2). In healthy plants (DSI 0), the xylem and pith were well-organized and structurally intact across all species (Figs. 2A–2C). The xylem exhibited clearly defined tracheid cells, and the pith appeared uniform, without signs of damage or abnormality. The resin canals (RC) were clearly visible, and the pith (P), located centrally within the stem, remained structurally undisturbed.

As infection progressed, increasing damage to xylem and pith tissues was observed, with species-specific variations in severity (Figs. 2D–2O). At DSI 1, P. massoniana exhibited slight discoloration in the xylem and early-stage resin accumulation (Fig. 2D), while P. elliottii × P. caribaea showed minimal tissue changes with slight yellowing near the pith (Fig. 2E). P. elliottii displayed only minor resin deposition around the pith, with no evident structural collapse (Fig. 2F).

By DSI 2, P. massoniana exhibited moderate tissue degradation, characterized by brown lesion formation and increased resin deposition around the xylem (Fig. 2G). P. elliottii × P. caribaea showed a distinct necrotic patch in the pith, indicating progressive damage (Fig. 2H), while P. elliottii displayed clear necrotic changes within the pith but maintained overall xylem integrity (Fig. 2I).

At DSI 3, P. massoniana demonstrated extensive xylem disruption, resin accumulation, and visible necrotic patches throughout the vascular tissue (Fig. 2J). P. elliottii × P. caribaea exhibited severe necrosis, with resin canals appearing blocked and structural collapse evident in the pith (Fig. 2K). P. elliottii, although showing localized necrosis, maintained relatively intact xylem structure compared to the other two species (Fig. 2L).

By DSI 4, P. massoniana displayed complete vascular collapse, widespread necrosis, and severe structural degradation (Fig. 2M). P. elliottii × P. caribaea showed near-total loss of vascular integrity, with extensive resin accumulation and pith decay (Fig. 2N). In contrast, P. elliottii exhibited severe necrosis but retained partial xylem structure, suggesting a comparatively slower rate of tissue degradation (Fig. 2O).

These findings indicate that the severity of vascular tissue damage correlates with species susceptibility, with P. massoniana exhibiting the most pronounced xylem and pith disruption, P. elliottii × P. caribaea showing intermediate damage, and P. elliottii maintaining better structural integrity, consistent with its higher resistance to B. xylophilus.

Susceptibility rate, disease severity index, and mortality rates

The progression of pine wilt disease (PWD) in the three pine species was evaluated based on susceptibility rate, disease severity index (DSI), and mortality rate over a 35-day period following inoculation with B. xylophilus. The effects of different inoculation doses were also analyzed to determine the influence of nematode density on disease development.

The susceptibility rate, defined as the percentage of symptomatic plants, increased progressively in all species throughout the observation period (Fig. 3). At lower inoculation doses (2,000 PWN per plant, Fig. 3A), P. massoniana exhibited a rapid increase in susceptibility, reaching 80% by 21 days post-inoculation (dpi), while P. elliottii × P. caribaea and P. elliottii showed a slower progression, with final susceptibility rates of approximately 90% and 100%, respectively, by 35 dpi. At higher inoculation doses (4,000 and 8,000 PWN per plant, Figs. 3B and 3C), all species showed a more rapid increase in susceptibility, with nearly all plants developing symptoms by 28 dpi. These trends across all inoculation doses indicated that P. massoniana was the most susceptible early in the infection process, but by the later stages (28–35 dpi), the susceptibility rates among the three species were comparable.

The progression of disease severity varied among species, with clear distinction observed in the distribution of plants across DSI categories (Fig. 4). At 7 dpi, the majority of plants remained in DSI 0 or 1, indicating minimal symptom development across all species. However, by 14 dpi, P. massoniana exhibited a substantial shift towards higher DSI levels, with many individuals reaching DSI 2 or 3, while P. elliottii × P. caribaea and P. elliottii retained a higher proportion of plants at lower DSI levels. By 21 dpi, P. massoniana had the highest proportion of plants in DSI 3 and 4, indicating severe wilting and chlorosis.

By 28–35 dpi, plants from all three species exhibited significant disease progression, but P. elliottii maintained a higher proportion of plants at DSI 2 or lower compared to the other two species, which had shifted predominantly to DSI 3 and 4. These results suggest that P. massoniana was the most susceptible to severe disease progression, followed by P. elliottii × P. caribaea, while P. elliottii exhibited the slowest disease development and retained a greater number of less severely affected individuals.

Mortality rates followed a similar trend to the DSI progression, with increasing mortality observed over time and at higher inoculation doses (Fig. 5). At 2,000 PWN per plant (Fig. 5A), P. massoniana exhibited the highest mortality rate by 35 dpi, while P. elliottii had the lowest. Increasing the inoculation dose to 4,000 and 8,000 PWN per plant (Figs. 5B and 5C) resulted in significantly higher mortality across all species, with nearly 100% mortality observed in P. massoniana and P. elliottii ×P. caribaea at 35 dpi.

Across all tested inoculation doses, P. massoniana consistently showed the highest mean susceptibility rate, disease severity index, and mortality rate. P. elliottii ×P. caribaea exhibited an intermediate response, while P. elliottii demonstrated greater resistance, showing slower symptom development, lower DSI scores, and reduced mortality, even at high inoculation doses. These results highlight the potential for utilizing P. elliottii in breeding programs aimed at enhancing resistance to PWD in pine plantations.

Nematode migration within the plant

To compare the migration and reproductive efficiency of B. xylophilus across different Pinus species, PWN density was assessed at the upper and lower sections of the inoculation site at various inoculation doses (Table 1; Fig. 6). The analysis revealed significant differences in PWN density among tree species (P < 0.05) and inoculation doses (P <  0.05), but no significant difference in the direction of PWN migration (P > 0.05), indicating that PWNs migrated equally in both upward and downward directions across all species and doses.

Regarding species differences, P. massoniana exhibited significantly higher PWN densities than P. elliottii × P. caribaea and P. elliottii (Fig. 6A). These results suggest that P. massoniana may be more favorable for PWN settlement compared to the other species, although the overall migration of PWNs into the stem was similar across species.

With respect to inoculation doses, PWN density increased significantly with higher inoculation doses (Fig. 6B). The highest PWN density was observed at the 8,000 PWNs per plant dose, followed by 4,000 and 2,000 doses. This dose-dependent increase in PWN density suggests that greater inoculation pressure leads to higher PWN migration and reproduction within the plant.

The interaction between tree species and inoculation dose was further explored by comparing PWN density in the upper and lower portions of the inoculation site (Figs. 6C, 6D). In the upper portion (Fig. 6C), significant differences in PWN density were observed at the 4,000 and 8,000 inoculation doses. P. massoniana exhibited the highest densities, followed by P. elliottii × P. caribaea and P. elliottii. Similarly, in the lower portion (Fig. 6D), significant differences were observed at the 4,000 and 8,000 doses, with P. massoniana showing the highest densities, followed by P. elliottii × P. caribaea and P. elliottii. These results indicate that PWNs migrate similarly in both directions across species, but species still exhibit varying levels of resistance at higher inoculation doses.