Trichoderma spp. isolates stimulate rice seedling growth of Sertani 13 variety

Trichoderma has become one of the most studied filamentous fungi to be used as a greener and more sustainable solution for improving the production and growth of numerous crops, due to its capability to form symbiotic associations with plants. This study aimed to evaluate the effect of Trichoderma isolates obtained from the rhizosphere of organic rice fields in Sukabumi, Indonesia, in enhancing rice germination and seedling growth. A laboratory experiment used a completely randomized design consisting of seed treatments of 21 Trichoderma isolates (T1-T21) and a control treatment without Trichoderma (C). The inoculation was employed to elucidate any potential effects of Trichoderma isolates. Results showed that five isolates, i.e., T5, T7, T9, T10, and T14 stimulated the highest seedling vigor index, root and shoot length, and fresh weight and dry weight. These findings exhibited the potential of these five isolates as plant growth-promoting fungi to improve rice seedling growth and contribute to our understanding of the role of symbiotic fungi in sustainable rice crop production.


INTRODUCTION
Rice (Oryza sativa L.) is an important cereal crop which serves as a staple food for over 50% of the global population, especially in Asia (Ng et al., 2015).However, there has been a lack of progress in the enhancement of rice productivity and expansion of cultivation land in the past twenty years (FAO, 2020).This is attributed to the growing scarcity of resources (land, water, and labor), as well as the ineffective utilization of agrochemical inputs, along with the escalating expenses associated with rice cultivation (Abdullah et al., 2021;Basu et al., 2021;Kumar et al., 2022;Prasad et al., 2017).Therefore, efforts must be made to increase rice production, and one innovative approach that should be considered due to its eco-friendly nature and low cost is the utilization of microbialbased inoculation (Akbari et al., 2023).Microbes can play crucial roles in enhancing rice growth by facilitating nutrient acquisition, influencing physiological processes and development, modulating gene expression, and suppressing phytopathogens without degrading the environment (Doni et al., 2022).Additionally, harnessing microbial-based inoculation could potentially lead to sustainable agricultural practices, ensuring food security for future generations while minimizing environmental impact (Wei et al., 2024).
For the past few decades, microbes have been scientifically examined and confirmed to play an important role in the preservation of soil health, as well as in the enhancement of rice productivity (Hanudin et al., 2018).Beneficial microorganisms have been used in sustainable agriculture for decades due to their potential to act as plant growth enhancers (Doni et al., 2014b;Yadav et al., 2017).The utilization of plant growth-promoting microorganisms has been empirically demonstrated to possess the capacity to enhance seed germination, enhance seed vigor, and promote seedling growth (Shahrajabian et al., 2021).Trichoderma is one of the plant growth-promoting microorganisms capable of enhancing the growth of rice plants (Doni et al., 2023).
Trichoderma (Hypocreales, Hypocreaceae; teleomorph Hypocrea) is a genus of mycotrophic filamentous ascomycete fungi that are widely isolated from rotting wood, bark, other fungi, construction materials, and mammals (Jaroszuk-ściseł et al., 2019).Trichoderma has been shown to be able to colonize plant roots in order to maintain symbiotic interactions with plants (Doni et al., 2017;Akbari et al., 2024).In exchange for providing Trichoderma with an appropriate environment and food, the host plant roots obtain favorable regulation of growth, yield, and stress tolerance (Alfiky & Weisskopf, 2021).
Trichoderma can be utilized as a bioinoculant for seed treatment in order to boost seedling growth (Pokhrel et al., 2022), or called plant growth-promoting fungi.Seed treatment emerges as a compelling method for applying antagonistic microorganisms, as other procedures require the utilization of larger quantities of propagules (Gava & Pinto, 2016).Seed treatment with beneficial microorganisms incurs relatively low application costs, does not induce alterations within the seed, and provides advantages to the crop during both germination and seedling growth phases (Cardarelli et al., 2022).
The efficacy of several Trichoderma isolates for enhancing rice seed germination has been documented in several studies.For instance, a study using a Malaysian rice variety MRQ74 demonstrated the positive effects of Trichoderma sp.SL2 on rice seedling growth and vigor, evidenced by notable increases in seedling shoot and root length (71% and 82%, respectively), shoot and root weight (47% and 153%, respectively), vigor index (91%), and germination speed (25%) compared to untreated control (Doni et al., 2014a).Furthermore, another study emphasized the efficacy of Trichoderma sp. 3 in enhancing the germination rate of the Junjuang rice variety by 21% and increasing the vigor index by 174% (Elita et al., 2023).Despite the documented benefits of Trichoderma in enhancing rice seed germination and seedling vigor, there is a notable gap in research regarding the influence of seed treatment with Trichoderma isolated from West Java, Indonesia on the growth of rice seedlings, especially in the context of Indonesian agricultural practices.Hence, the objective of this study was to assess the impact of seed treatment with 21 Trichoderma isolates, obtained from the rhizosphere of a rice field in Sukabumi, West Java, Indonesia, on rice seed germination and subsequent seedling growth, aiming to evaluate the promotion of rice seedling growth by Trichoderma.

Research site and experimental design
The research was carried out at the Applied Microbiology and Plant Pathology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jatinangor.The research was conducted from December 2021 -February 2022.
In this experiment, a completely randomized design was employed, consisting of 22 treatments and three replications.The treatments were 21 isolates of Trichoderma (T) and one control (C).The seed treatments evaluated for this study are rice seeds soaked in distilled water as the control group (C) and rice seeds soaked in spore suspension of Trichoderma isolated from Nusantara Organic SRI Center (NOSC), Sukabumi, West Java (T).For each treatment, the seeds were soaked for 24 hours.The data were measured quantitatively.Rice germination parameters were measured including germination percentage and vigor index.Rice seedling growth parameters consisted of root and shoot length, fresh weight, and dry weight.

Fungal cultures and preparation of inocula
Trichoderma spp. was isolated from soil samples collected from organic rice fields in Sukabumi using a multilevel dilution technique in the Applied Microbiology and Plant Pathology Laboratory, Universitas Padjadjaran.The process involved serial dilution of a 10 g soil sample with 100 mL distilled water, creating homogenized suspensions.Before the soil became a precipitate, a sterile pipette was used to collect 1 mL of the suspension, which was then diluted into 9 mL of distilled water.The dilution process was performed three times, resulting in a dilution rate of 10 −1 to 10 −5 g of soil per mL.A volume of 100 μL of the solution was pipetted out and transferred using the pour plate technique onto the prepared potato dextrose agar (PDA) medium.The agar was then incubated at 26 °C until fungal colonies formed.Subsequently, the isolates underwent purification and were moved to a new petri dish containing fresh PDA medium (Mishra et al., 2019).
After a seven-day incubation at 26°C, twenty-one purified isolates of Trichoderma (T1-T21) were used to make a spore suspension.The spores were obtained from plates with the addition of 10 mL of sterile water.Subsequently, the spores were directly moved to an erlenmeyer flask filled with sterilized distilled water.The spore concentration was adjusted to 10 7 spores mL -1 based on measurements obtained using a hemocytometer.The following formula was used to calculate spore density (Anhar et al., 2018): Spore density = (number of conidia × 5 ×dilution factor)/(haemocytometer volume)

Rice seed inoculation and seedling preparation
In this research, the Indonesian Sertani 13 rice (Oryza sativa L.) cultivar was used.The seeds were sterilized by soaking them in 70% ethanol for 30 minutes and then rinsing them with sterile distilled water.For the control treatment, 300 seeds were soaked for 24 hours in sterile, distilled water.
Trichoderma-treated seeds (300 seeds per treatment) were soaked for 24 hours in a spore suspension.The seedlings were then grown for a duration of five days within sterile Petri dishes, wherein the growth media consisted of filter paper moistened with distilled water (Doni et al., 2017).

Observations
The germination percentage refers to the mean proportion of seeds that undergo germination within five days.The seeds were cultivated on filter paper in petri dishes under controlled conditions at a temperature of 26°C.The total amount of normal seedlings was collected, which indicated by rice seedlings with essential structures (seminal roots, mesocotyl, coleoptile, cotyledon, and primary leaf).The germination percentage was determined using the subsequent formula: Germination (%) = (number of normally germinated seeds)/(number of seeds) × 100 The calculation of a seed vigor index (SVI) was performed using the formula provided by Abdul-Baki and Anderson (1973): Seed vigor index = germination (%) × seedling length (shoot length + root length) (cm) The lengths of the roots and shoots were assessed on the fifth day after the radicle had emerged.A total of 20 seedlings were randomly chosen from each treatment, and their lengths were measured using a ruler.The measurement of root length was conducted by determining the distance between the apex of the primary root and the base of the hypocotyl.The measurement of shoot length was conducted by determining the distance between the base of the primary leaf and the base of the hypocotyl.The fresh weight and dry weight (in milligrams) of seedlings were measured using a digital scale.The seedlings were subjected to drying in an oven at 65°C for 48 hours, following which their dry weight was measured (Mishra et al., 2019).

Statistical analyses
All data were statistically examined using one-way analysis of variance (ANOVA).The Duncan Multiple Range Test (DMRT) at p<0.05 was used to separate mean values for variables with significant effect.

Seed germination and vigor
The germination percentage and seedlings vigor differed significantly among treatments (p ≤ 0.05) (Table 1).The germination percentage of rice seeds in this study ranged from 66% to 94%.While the seed treatment with Trichoderma isolates did not exhibit a significantly higher germination percentage compared to the control, certain Trichoderma isolates showed a tendency to enhance vigor index values.Seed treatment with T1 showed a notable seed germination percentage (94%); however, this result did not demonstrate a significant difference compared to C, T2, T4, T8, T12, T14, T17, T18, T19, and T20 treatments, indicating comparable effects across these Trichoderma isolates and control.Meanwhile, T10 exhibited the lowest germination percentage among the treatments.Among Trichoderma isolates, T14 demonstrated the highest seed vigor index (1337.70),in comparison to the other treatments.Furthermore, findings suggest that isolates T4, T5, T7, T9, T13, T15, T16, T17, and T20 also displayed higher vigor index values compared to the control treatment, underscoring their potential as promising isolates for improving rice seedling vigor.Conversely, the control treatment (C) exhibited the lowest vigor index (759.29).Note: The treatments T1-T21 (inoculated with native Trichoderma isolates).Values followed by the same letters in the same column are not significantly different according to DMRT (p<0.05).
The presence of high seed vigor is correlated with the capacity to enhance growth and productivity within the realm of agricultural production (Han et al., 2014).The findings of our study align with those of a prior investigation conducted by Doni et al. (2014b), who observed an enhancement of germination percentage and seedling vigor of rice with the application of seven Trichoderma isolates.In another study, the inoculation of T. harzianum and T. minutisporum increased the germination percentage, vigor index, and germination speed of local Indonesian rice varieties (AA75, Mikonga, Batang Sungkai, Saganggam) (Anhar et al., 2021).The enhanced germination and seed vigor seen in this study may be attributed to the presence of several phytohormones like auxin, cytokinin, zeatin, and gibberellin, which are released by Trichoderma isolates (Osiewacz, 2002;Swain et al., 2018).Furthermore, seed treatment with the promising Trichoderma isolates can stimulate the production of gibberellin which activates germination by promoting enzyme activity, such as amylase, which facilitates starch metabolism in rice seedlings (Piri et al., 2019).In addition, the type of Trichoderma strain and numerous external stimuli influence the production of growth regulators (Nieto-Jacobo et al., 2017).However, in this study, not all Trichoderma isolates are able to enhance germination.Santos et al. (2020) reported a comparable opposing outcome, wherein Trichoderma was discovered to have a suppressive impact on the germination percentage of Handroanthus serratifolius.The variation in germination outcomes can be attributed to the isolatespecific effect, as certain isolates have the potential to inhibit germination (Machado et al., 2015).This inhibition may result from the presence of phytotoxic secondary metabolites, such as trichothecenes produced by Trichoderma at specific concentrations et al., 2020).
The observed effects on shoot and main root length in rice seedlings induced by Trichoderma isolates mainly may be attributed to the synthesis of growth-stimulating hormones and secondary metabolites.The observed enhancements in shoot and main root length align with findings from previous research, establishing a consistent pattern across diverse crops such as wheat (Saadaoui et al., 2023), Mongolian pine (Halifu et al., 2019), and tomato (Singh et al., 2014).
Beneficial root-colonizing microorganisms like Trichoderma that release IAA can potentially influence the plant's endogenous IAA when employed in seed treatment (Backer et al., 2018).IAA is essential for root and shoot growth in plants (Halifu et al., 2019).Apart from plant hormones, Trichoderma was discovered to produce harzianolide, a secondary metabolite capable of influencing the early phases of seedling growth by increasing root length and root tips and regulating overall root development (Cai et al., 2013).These findings not only contribute to the growing body of knowledge on seed treatment with Trichoderma but also suggest its potential application as a sustainable strategy for enhancing seedling growth in rice and other crops.

CONCLUSIONS
Five out of 21 Trichoderma isolates, i.e., T5, T7, T9, T10, and T14 were promising bioinoculants as shown by positive effects on higher root, shoot length, fresh and dry weight of rice seedlings.The germination percentage of Trichoderma-treated seeds was not significantly different from the control treatment, therefore, evaluation of seedling growth variable is important in the future.Further research is needed to investigate the performance of Trichoderma-treated seeds under actual field conditions, considering factors such as soil types, climatic conditions, and agronomic practices.

Table 1 .
Effects of Trichoderma on germination percentage and seedling vigor index of rice seedlings.

Table 2 .
Root length and shoot length of rice seedlings with Trichoderma inoculation.

Table 3 .
Fresh weight and dry weight of rice seedlings with Trichoderma inoculation.