Synergistic Effects of a Mycorrhizal Consortium on Growth, Morphophysiological Traits, and Nodulation Efficiency in Dalbergia latifolia Roxb

1. Introduction

Dalbergia latifolia Roxb., commonly known as Indian rosewood, is a highly valued timber species from the Fabaceae family. Despite its commercial significance, it is sparsely distributed across tropical and subtropical habitats [1]. In addition to its demand for timber, D. latifolia is known for its medicinal properties, particularly its tannin content, which is used in the treatment of leprosy and parasitic infections [2]. Classified as ‘Vulnerable’ by the International Union for Conservation of Nature (IUCN) and listed under Appendix II of CITES, the species faces a decline in population due to factors such as overharvesting, illegal trade, habitat destruction, and environmental degradation [1,3]. To ensure its survival, conservation efforts and artificial regeneration within its native habitats are essential. Large-scale cultivation and afforestation initiatives will also play a key role in sustaining D. latifolia [4].

Arbuscular mycorrhizal (AM) fungi, beneficial soil microbes that form symbiotic relationships with plants, have shown potential in enhancing nutrient uptake, particularly phosphorus (P), zinc (Zn), and copper (Cu). These fungi also improve plant resilience to biotic and abiotic stresses [5]. In addition to their role in nutrient uptake, AM fungi contribute to soil structure, nutrient cycling, and ecosystem sustainability [6]. By optimizing water and phosphorus absorption, AM fungi promote plant growth [7], and their use as biofertilizers is becoming increasingly relevant for enhancing plant health and productivity.

This study investigates the impact of AM fungi on the growth performance of Dalbergia latifolia and assesses their potential in supporting sustainable cultivation practices. Previous research has documented the positive effects of AM fungi on plant growth, with studies highlighting their role in micropropagation [8] and focusing on medicinal plants [9]. Moreover, the beneficial impact of AM fungi on nutrient uptake and root development has been reported in various species [10]. The present study extends these findings to Dalbergia latifolia, exploring the broader potential of AM fungi for improving the cultivation of this important timber species.

Additionally, Dalbergia latifolia has gained attention for its potential role in agroforestry systems. A comprehensive tree database supports the selection of suitable species for agroforestry, further emphasizing the relevance of AM fungi in promoting the sustainable cultivation and ecological benefits of this species [11].

2. Materials and Methods

2.1. Study Site and Experimental Conditions

The nursery trials were conducted at the forest nursery of the Institute of Wood Science and Technology (IWST), Bengaluru, Karnataka, India (latitude 12°51′30.7″ N; longitude 76°84′83.9″ E). The study area, located in the Eastern Dry Zone, receives an average annual rainfall of approximately 900 mm, with temperatures ranging from 35°C in the summer to 8°C in the winter.

2.2. Planting Material Collection and Seed Treatment

Mature seeds of Dalbergia latifolia were collected during the January seed-bearing season from a candidate plus tree in the Bhadra Tiger Reserve, Chikkamagaluru Forest Division, Karnataka, India. The seeds were assessed for health and viability according to the guidelines of the International Seed Testing Association [12]. Prior to sowing, the seeds were surface-sterilized with a 2% Bavistin solution, thoroughly rinsed, and soaked in cold water for 24 hours to enhance germination.

2.3. Nursery Experiment and Experimental Design

Seeds of Dalbergia latifolia were germinated in a sand bed containing heat-sterilized sand (121°C for 1 hour). At 30 days post-germination, uniform, healthy seedlings, free from diseases and pests, were selected and transplanted into 15 × 12-inch plastic pots filled with an autoclaved mixture of red soil and sand (1:1, v/v). Arbuscular mycorrhizal (AM) fungal inoculation was performed by applying 10 g of inoculum (containing spores and hyphal fragments) 2 inches away from the stem and 5 inches below the root zone during transplantation. Each treatment was replicated three times, resulting in a total of 30 plants for the study. The plants were maintained under nursery conditions with regular irrigation and proper care throughout the experimental period. Control plants were kept without AM fungal inoculation.

The experiment followed a randomized block design (RBD) with four treatments and three replications:

• T1 (Control): D. latifolia without AM fungal inoculation.
• T2: D. latifolia inoculated with Glomus mosseae.
• T3: D. latifolia inoculated with Glomus fasciculatum.
• T4: D. latifolia inoculated with Glomus leptotichum.

Growth parameters were recorded at 3-, 6-, 9-, and 12-month intervals post-inoculation.

2.4. Arbuscular Mycorrhizal (AM) Fungal Inoculum

The AM fungal species used in this study were Glomus mosseae, Glomus fasciculatum, and Glomus leptotichum. The inoculum was obtained from Dr. D. J. Bagyaraj, NASI Honorary Scientist & Chairman of the Centre for Natural Biological Resources and Community Development (CNBRCD), Bengaluru, Karnataka, India. The inoculum consisted of a sand-soil mixture containing AM fungal spores, mycelia, and colonized root fragments.

2.5. Estimation of AM Fungal Root Colonization

AM fungal colonization was assessed at 3-, 6-, and 12-month intervals following the method described by Phillips and Hayman [13]. Root samples were randomly collected, cut into 1 cm segments, and cleared by autoclaving with 10% KOH at 108 kPa for 15 minutes. After neutralizing the alkalinity with 10% HCl, the roots were stained with 0.03% trypan blue in lactoglycerol. The stained root fragments were examined under a microscope for AM fungal structures, and the percentage of root colonization was calculated using the following formula:

% Root Colonization = (Total number of root bits examined / Number of root bits with AM fungal structures) × 100.

2.6. Enumeration of AM Fungal Spores

Extrametrical chlamydospores were quantified using the wet sieving and decanting method described by Gerdemann and Nicolson [14] (1963). A 100 g representative soil sample from each treatment was suspended in water, stirred thoroughly, and passed through a series of sieves (1000, 300, 205, 105, and 45 µm). The material retained on the bottom two sieves was transferred to a nylon mesh with an equivalent pore size. Spores retained on the mesh were then transferred to Petri dishes and counted under a microscope.

2.7. Morphological and Physiological Evaluations

The following growth and physiological parameters were assessed: plant height (cm), average leaf length (cm), average leaf width (mm), shoot length (cm), shoot width (mm), root length (cm), root width (cm), number of nodules, and total chlorophyll content in fresh leaves, measured using the method described by Arnon [15] (1949).

2.8. Data Analysis

Experimental data were analyzed using one-way analysis of variance (ANOVA) in Microsoft Excel software. Treatment means were compared and ranked using Duncan’s Multiple Range Test (DMRT) at a 5% level of significance [16].

3. Results

3.1. AM Fungal Colonization

The effects of AM fungal inoculation on the growth and physiological parameters of Dalbergia latifolia were assessed over a 12-month period. Statistically significant differences (p ≤ 0.05) were observed in various growth parameters between AM fungal-inoculated plants and the control group (T1), as shown in Table 1. The percentage of AM fungal colonization, the number of vesicles, arbuscules, and sporulation in the rhizosphere increased progressively over time. The micropropagated plants inoculated with Glomus fasciculatum (T3) showed 40% colonization, while the normal plants inoculated with the same species exhibited 35% colonization. The micropropagated plants demonstrated more prominent sporulation, which positively correlated with the number of vesicles and arbuscules, indicating higher mycorrhizal activity in the micropropagated plants.

3.2. Growth Parameters

Growth improvements were evident in all AM fungal-inoculated plants compared to the control group. The plants inoculated with Glomus mosseae (T2) showed the highest plant height throughout the study, reaching a maximum height of 112 cm at 12 months. Both Glomus fasciculatum (T3) and Glomus leptotichum (T4) exhibited substantial height increases, significantly outperforming the control group. This growth pattern was statistically validated through ANOVA and F-tests, which showed significant differences at the 1% and 5% levels.

3.3. Leaf Parameters

Leaf length and leaf width improved significantly under AM fungal inoculation. At the end of the study, G. leptotichum (T4) recorded the longest leaves (6.733 cm), followed by G. fasciculatum (T3) at 6.433 cm. G. mosseae (T2) showed the widest leaves at earlier stages (3 and 6 months), but G. fasciculatum (T3) achieved the largest leaf width (5.1 mm) at 12 months.

3.4. Shoot Growth

Shoot length followed a similar upward trend. At 3 months, plants inoculated with G. fasciculatum (T3) had the longest shoots (16.33 cm). However, by 9 and 12 months, G. mosseae (T2) exhibited the highest shoot length, reaching 76 cm by 12 months, followed by G. fasciculatum (T3) and G. leptotichum (T4). Shoot width also increased, with G. fasciculatum (T3) and G. leptotichum (T4) achieving the largest shoot width (0.87 mm) by the study's conclusion.

3.5. Root Growth

Root length and root width were significantly greater in AM-inoculated plants compared to the control group. G. fasciculatum (T3) plants exhibited the longest roots, followed by G. leptotichum (T4) and G. mosseae (T2), while the control plants consistently displayed the shortest roots. The improvement in root development was mirrored in root width, with G. fasciculatum (T3) and G. leptotichum (T4) showing significant increases in root diameter.

3.6. Root Nodulation

Root nodulation was significantly influenced by AM fungal inoculation. G. mosseae (T2) consistently produced the highest number of nodules, followed by G. fasciculatum (T3) and G. leptotichum (T4), while the control group showed the fewest nodules, indicating enhanced nitrogen fixation in AM-inoculated plants.

3.7. Chlorophyll Content

Chlorophyll content, an indicator of photosynthetic efficiency, was significantly higher in AM fungal-inoculated plants at all time intervals. G. mosseae (T2) exhibited the highest chlorophyll content, reaching a peak of 60.33 at 9 months and maintaining a value of 57 at 12 months. Both G. fasciculatum (T3) and G. leptotichum (T4) showed significant increases in chlorophyll content compared to the control group, which displayed the lowest chlorophyll content throughout the study.

These results collectively indicate that AM fungal inoculation significantly enhances growth, root development, chlorophyll content, and nitrogen fixation in Dalbergia latifolia, underscoring the beneficial effects of mycorrhizal symbiosis on plant health and productivity.

4. Discussion

The findings of this study demonstrate that the inoculation of Dalbergia latifolia with arbuscular mycorrhizal (AM) fungi significantly boosts growth parameters, aligning with similar observations in other medicinal plants. For instance, improved growth in Kalmegh was reported by Arpana and Bagyaraj [17] as a result of treatment with AM fungi under phosphorus fertilization. The positive effects of AM fungi on nutrient uptake and chlorophyll content observed in this study are consistent with the findings of Zuccarini [18], who reported that mycorrhizal infection enhances nutrient uptake and chlorophyll content in lettuce under saline irrigation. Furthermore, the positive influence of AM fungi on root development in Dalbergia latifolia mirrors the results from Chiramel et al. [19], who documented similar improvements in root growth in Andrographis paniculata when inoculated with AM fungi. Furthermore, this study highlights the contribution of AM fungi in improving nutrient uptake in Dalbergia latifolia, as shown by Rajeshkannan et al. [20], who reported increased growth and nutrient absorption in this species under tropical nursery conditions with the use of bioinoculants. The influence of AM fungi on soil properties is also well-supported in literature. Research by Syam Prasad et al. [21] highlighted the ability of these fungi to positively influence soil physio-chemical properties, which, in turn, fosters plant growth. This view is also supported by Wahab et al. [5], who showed that AM fungi help regulate plant growth and productivity, especially under biotic and abiotic stress conditions. These findings align with the conclusions of Martin and Heijden [6], who emphasized the broader ecological and agricultural benefits of mycorrhizal symbiosis, particularly in promoting nutrient cycling and improving soil structure. The potential of AM fungi to enhance plant health and improve soil properties emphasizes their role in sustainable agriculture. Furthermore, the work of Gerdemann and Nicolson [14] on spore extraction techniques underscores the importance of understanding the AM fungal life cycle for efficient inoculation, a critical aspect for future studies. Beyond the ecological and economic value of Dalbergia latifolia as discussed by Arunkumar et al. [1], the integration of AM fungi could play a key role in supporting the conservation of this species by promoting its growth and regeneration, as noted by Sasidharan et al. [3].

The positive outcomes observed in this study further highlight the necessity of sustainable management practices for supporting the growth and conservation of Dalbergia latifolia. Moreover, Dalbergia latifolia holds significant promise for agroforestry systems, as indicated by Orwa et al. [11], reinforcing the value of integrating AM fungi into these systems to boost productivity and maintain ecological balance. Overall, the results from this study stress the vital role of AM fungi in enhancing the growth and health of Dalbergia latifolia and similar species, as demonstrated in several other studies by Smith and Read [22] and Janos [23]. The application of AM fungi in cultivating medicinal and forest species has considerable potential for improving productivity and fostering ecological sustainability.