Efficacy of Bioinoculant Trichoderma asperelloides in Promoting Plant Growth in Grevillea robusta

Short Communication

Forest nurseries play a crucial role in producing quality planting stock (QPS) for afforestation and reforestation programs. Plantation managers primarily focus on achieving early and vigorous QPS to ensure successful establishment. However, seedling growth and development in forest nurseries are largely influenced by soil nutrient availability and biotic constraints. While application of chemical fertilizers and pesticides is a common approach to promoting vigorous QPS, this may pose significant environmental concerns, including pollution, soil degradation, and potential health risks to both humans and ecosystems. Therefore, nursery managers need eco-friendly alternatives to ensure sustainability. Trichoderma species are integral to plant health, functioning as growth enhancers and biocontrol agents, inducers of systemic resistance against a broad spectrum of biotic threats, and effectively suppress and kill destructive pathogens. Furthermore, these fungi play a vital role in alleviating abiotic stresses such as extreme pH, drought, cold, and salinity, underscoring their significance in sustainable crop production (see reviews by Harman et al., ² Druzhinina et al., ¹ and Mukherjee et al. ³ ) Notably, the application of Trichoderma species has been extensively studied for enhancing growth performance in agricultural crops and, to a lesser extent, in forestry species, with successful evaluations in Acacia mangium in Malaysia, ⁴ Pinus sylvestris var. mongolica in China ⁵ and Handroanthus serratifolius in Brazil. ⁶

In India, Grevillea robusta is a valuable agroforestry species with significant potential for supplying raw materials to the paper, pulp, and wood-based industries. Due to its economic and ecological importance, large-scale cultivation of G. robusta has been expanding. However, nursery-grown seedlings often suffer from poor growth and foliage browning, which may be attributed to various factors, including diseases and abiotic stress. Therefore, this study aimed to assess the efficacy of Trichoderma in enhancing the production of QPS of G. robusta.

A Trichoderma strain (T₁), which demonstrated strong biocontrol potential against several important forestry pathogens (unpublished data), was selected as a bioinoculant. Since Trichoderma species exhibit high morphological similarity, leading to potential misidentifications, molecular identification was performed using sequence analysis of the translation elongation factor 1-alpha (tef1) region, as recommended. ⁷ Genomic DNA was extracted from a 10-day-old fungal colony of strain T₁ grown on potato dextrose agar (PDA) using a mini-DNA extraction protocol. ⁸ The tef1 region was amplified using the primer pair EF1 and EF2, ⁹ and sequencing was outsourced to Biokart India Pvt. Ltd. (Bengaluru, India) using an Applied Biosystems sequencing platform following the manufacturer’s protocol. A BLAST search (https://www.ncbi.nlm.nih.gov) of the resulting tef1 sequence (GenBank PV266533) revealed >99% similarity to T. asperelloides (Viride clade) GenBank accessions.

To further confirm the identification, maximum likelihood (ML) analysis was performed using the W-IQ-TREE web server. ¹⁰ The analysis included the tef1 sequence generated in this study along with authentic and reference sequences from 112 species in the Viride Clade. Sequence alignment was conducted using MAFFT v7 (https://mafft.cbrc.jp/alignment/server/) ¹¹ . An ultrafast bootstrap (UFBoot) analysis with 10000 alignments ¹² and an SH-like approximate likelihood ratio test (SH-aLRT) with 10000 replicates ¹³ were conducted to enhance the accuracy and minimize bias in branch support values (http://www.iqtree.org/doc/Home). The ML alignment comprised 170 sequences with 724 columns, 594 distinct patterns, 479 parsimony-informative sites, 65 singleton sites, and 180 constant sites. The best-fit model selected based on the lowest Bayesian Information Criterion (BIC) score was TN+F+I+G4. The log-likelihood of the best ML tree was −19422.6870 (s.e. 599.4149), with 338 free parameters, a BIC score of 41071.0335, an AIC score of 39521.3740, an AICc score of 40116.6052, a total tree length of 11.4513, and a sum of internal branch lengths of 4.6642. Strain T₁ from this study formed a well-supported clade (SH-aLRT = 85, UFBoot = 97) with T. asperelloides accessions, including the type culture CBS 125398, while T. asperellum and T. yunnanense clustered as distinct yet closely related species (Fig. 1). ¹⁴ suggested that T. asperelloides is morphologically indistinguishable from T. asperellum and requires DNA sequence analysis for reliable and accurate identification.

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Fig. 1.

Phylogenetic tree generated from maximum likelihood analysis of tef1 sequence data of members of the Viride clade. Statistical support values are indicated at the nodes (SH-aLRT/UFBoot). The tree is rooted to Protocrea pallida CBS 121552. Ex-type cultures are denoted by a superscript “T”. The strain from this study is highlighted in red.

In nursery trials, a total of 300 G. robusta seedlings were selected, with 150 designated as control (C) and 150 treated with T. asperelloides. A spore suspension of T. asperelloides was prepared in sterile distilled water and adjusted to a concentration of 10⁸ spores mL⁻¹ using a hemocytometer. Before planting, seedlings were dipped in the spore suspension for 30 minutes, while control seedlings were dipped in sterile distilled water. Quarterly observations were recorded for key seedling growth parameters, including shoot length (SL, cm), collar diameter (CD, mm), number of leaves per plant (NoL), root length (RL, cm), and number of roots (NoR). Statistical analysis revealed significant differences (p < 0.05, Table 1) in SL, CD, RL, NoL, and NoR between Trichoderma-treated and control groups (Fig. 2). In addition to promoting growth, no foliage browning was observed in Trichoderma-treated seedlings, suggesting its potential application in G. robusta nurseries. However, further research is needed to determine the underlying causes of foliage browning and to elucidate the mechanisms by which T. asperelloides mitigates this issue.

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Fig. 2.

Growth performance of G. robusta: (a) control root, (b) T. asperelloides-treated root, (c) entire seedlings (control), and (d) T. asperelloides-treated seedlings (treatment).

Trichoderma is a versatile fungal genus that plays multiple ecological roles in the rhizosphere. It enhances plant growth in natural and agricultural systems by producing bioactive compounds and solubilizing soil nutrients. In addition, Trichoderma helps plants adapt to stressful conditions, making it a valuable ally in sustainable agriculture. ¹⁵ This study highlights the potential of T. asperelloides as a growth promoter in G. robusta, enhancing its resilience and development. Future research should focus on its mechanism of action, as well as testing and optimizing its applications in other forestry species. Exploring its synergistic interactions with other beneficial microbes could further enhance sustainable forestry practices.