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Agricultural Science Digest, volume 42 issue 6 (december 2022) : 741-746

Screening of Plant Growth Promoting Fungi (PGPF) for Sustainable Cultivation of Tulaipanji, an Endemic Aromatic Rice Variety of Uttar Dinajpur, West Bengal, India

Monalisha Sarkar1, Zerald Tiru1, Ayon Pal2, Parimal Mandal1,*
1Mycology and Plant Pathology Laboratory, Department of Botany, Raiganj University, Uttar Dinajpur, Raiganj-733 134, West Bengal, India.
2Microbiology and Computational Biology Laboratory, Department of Botany, Raiganj University, Uttar Dinajpur, Raiganj-733 134, West Bengal, India.
Cite article:- Sarkar Monalisha, Tiru Zerald, Pal Ayon, Mandal Parimal (2022). Screening of Plant Growth Promoting Fungi (PGPF) for Sustainable Cultivation of Tulaipanji, an Endemic Aromatic Rice Variety of Uttar Dinajpur, West Bengal, India . Agricultural Science Digest. 42(6): 741-746. doi: 10.18805/ag.D-5561.
Background: Rice is one of the major staple crops of human population. Worldwide escalating human populace exerts pressure to increase rice production. To fulfill the demand, the injudicious application of chemical fertilizers is posing grave threat to the environment together with the entire living population. A suitable replacement of these harmful fertilizers is needed in modern agriculture to keep the biodiversity undisturbed. The main objective of the study is to screen and explore indigenous plant growth promoting fungi which can replace the conventional fertilizers.

Methods: Rhizospheric soil of different rice cultivating fields were collected in the year 2018. Different in vitro assays of the fungal isolates obtained from the soil were performed to check their plant growth promotion capabilities.

Result: 20 different indigenous fungal isolates were studied to evaluate their ability as PGPF for organic cultivation of Tulaipanji, an endemic aromatic rice variety cultivated in Uttar Dinajpur, West Bengal, India. Trichoderma harzianum TaK12 and Trichoderma aureoviride TaN16 were found to enhance maximum shoot and root length of rice plant. These two isolates also showed efficacy in phosphate solubilization. Trichoderma aureoviride TaN16 together with Penicillium citrinum PcK10 and Aspergillus niger AnK1 produced high amount of IAA. The results obtained from the study clearly indicate that these plant growth promoting fungi (PGPF) possess the ability to boost up plant growth. 
Rice is the main staple crop that contributes to fulfill the food needs of St rikeout half of the human population across the globe. The increasing population of the earth puts pressure to escalate the rice production (Mahajan 2020). Excess fertilizers are applied in rice cultivation fields to encounter the escalating pressure of high production (Polthanee 2021). This results in negative impact on both the environment and on all living beings (Habibah 2011). In this perspective, an alternative to traditional fertilizers is urgent requisite to strengthen agricultural production in organic approaches. Plant growth promoting fungi (PGPF) due to their eco-friendly nature can be used in organic agriculture practice in place of these traditional inorganic fertilizers. The rhizosphere is inhabited by a large number of microbes that succor in plant growth and development and sustain soil fertility (Mendes et al., 2013). They may be free-living in the soil, epiphytic when residing on the root surface or endophytic dwelling inside the roots (Hossain et al., 2017). PGPFs are reported to synthesize plant hormones like IAA, solubilize soil phosphorus, produce low molecular weight volatile compounds called microbial volatile organic compounds (mVOCs), thus assisting in plant growth and development (Jogaiah et al., 2013). The PGPF also help in seed germination and seedling vigor, root and shoot growth, augmenting photosynthetic efficacy and improving crop-yield (Contreras-Cornejo et al., 2011).
 
PGPF is a heterogenous group of non-pathogenic, non-symbiotic, saprotrophic fungi that mainly belong to the phylum Ascomycota (Aspergillus, Fusarium, Penicillium, Phoma, Talaromyces, Trichoderma), Basidiomycota (Limonomyces, Rhizoctonia) and Zygomycota (Mucor, Rhizopus(Muslim et al., 2019). The property of PGPF for enhancing the growth of plants make them suitable for organic based agriculture and can be a new innovative approach in organic agriculture with less dependence on traditional inorganic fertilizers. In the present study, this approach is considered for organic cultivation of Tulaipanji, a renowned endemic aromatic rice variety of Uttar Dinajpur, West Bengal, India.
Collection of rhizospheric soil
 
Rhizospheric soil adhering to root of rice plants was collected in sterile polythene bags from different rice cultivating fields of Uttar Dinajpur, West Bengal in the year 2018. The samples were brought to Mycology and Plant Pathology Laboratory, Raiganj University.
 
Isolation of the fungal isolates
 
Isolation of indigenous soil borne fungi were executed through serial dilution method as demonstrated by Johnson and Curl (1972). In this method, 1 gm soil was dissolved in 10 ml of sterile double distilled water aseptically to make soil concentration 0.1 g/ml (10-1). A serial dilution in the range of 10-3 to 10-5 was prepared with sterilized double distilled water. 20 ml of potato dextrose agar (PDA) amended with Monocef antibiotic (2.5 mg/ml) was poured aseptically in each sterile Petridish and allowed to solidify for 1 hr. Then 1.0 ml of soil suspension from dilution 10-4 and 10-5 were poured into the Petridishes separately under aseptic condition and spread uniformly, followed by sealing the Petridishes with parafilm. The Petridishes were incubated at 28°C for 7 days. Different fungal colonies started to appear into the Petridishes after 3-7 days of incubation period. These colonies were picked up carefully and transferred on freshly prepared PDA slants and PDA plates, marked with proper labelling and incubated at 28±2°C for 7 days. The PDA slants were then stored at -4°C for future use.
 
Preparation of fungal inoculum
 
Spore suspension
 
Fungal isolates were multiplied in 250 ml Erlenmeyer flask containing 200 ml Potato dextrose broth (PDB) at 23±2°C for 10 days. Then the culture broth was centrifuged at 10,000 rpm for 10 mins. The pellets were suspended in sterile distilled water and washed repeatedly for 3-4 times. The washed fungal pellet was made into a turbid solution with sterile distilled water. The OD of the solution was adjusted to 0.45 at 610 nm to obtain 1×108 cfu ml-1 (Niranjana, Umesha et al., 2006).
 
Maize granules inocula (MGI)
 
One kilogram of maize grains was crushed into granules using mixer grinder for 2-3 minutes. Then the granules were semi-boiled for 5 minutes and air dried. The air-dried maize granules were amended with calcium carbonate (2%) and calcium sulphate (1%) at pH 6.48. Then mixed granules were filled (1/4th) in 500 ml Erlenmeyer flask, plugged with non-absorbent cotton, wrapped with paper and autoclaved. The autoclaved flasks were inoculated with mycelia discs (5 mm) obtained from the actively growing margins of 7 days-old culture of fungal isolates and incubated at 25°C. After 12-15 days of incubation period, completely colonized maize granules were used as maize granules inocula (MGI) and stored at -4°C until further studies.
 
Priming of seed with mycoflora isolates 
 
Rice seeds were surface sterilized with 0.1% mercuric chloride for 2-3 minutes and then rinsed 3-4 times with sterile distilled water. Then average 50 seeds were treated with 10 ml of conidial suspension (108 cfu ml-1) of fungal isolates. Three replicates were maintained for each treatment. In another set of experiment, surface sterilized 50 rice seeds were coated with MGI of different fungal isolates in sterile potting soil in the ratio of 5:95 (5%), 10:90 (10%) and 20:80 (20%). Treated seeds were kept at normal temperature for germination and growth observation. Seeds treated with sterile distilled water St rikeout served as untreated control. After 10 days, germination percentage root length and shoot length were recorded and vigor index was calculated following the method of Abdul-Baki and Anderson (1973).
 
Vigor index (VI) = Seed germination (%) × [Mean of root length + Mean of shoot length]
 
In vitro assay of indole acetic acid (IAA) production
 
Indole acetic acid (IAA) production was measured following standard methods with some modification (Bric et al., 1991). Fungal isolates were cultured on PDA media and incubated at 37°C for 7 days. After 7 days of incubation, fungal discs (5 mm) from culture media of each isolate were transferred to PDA broth amended with 500 µg/ml tryptophan as the precursor of IAA production and incubated in a shaker incubator at 28±2°C with rotation of 120 rpm for 3-5 days. Control set was maintained without tryptophan. Fully grown cultures were centrifuged at 10,000 rpm for 10 minutes. Then 1 ml of the supernatant was mixed with 4 ml of the Salkowski reagent (50 ml of 35% of HClO4, 1 ml of 0.5 m FeCl3 solution) followed by addition of 2-3 drops of 10 mM orthophosphoric acid and kept in dark for colour formation. Three replicates for each isolate were maintained. Appearance of pink color in test tubes indicated the production of IAA.
 
IAA production was expressed by + and - sign.
 
In vitro assay of phosphate solubilization
 
The phosphate (P) solubilizing activity of fungal isolates was performed on Pikovskaya’s solid agar medium amended with Rose Bengal at 10 ml/L as described by Johnson (1959) with some modifications. After incubation for seven days at 28°C, the formation of clear zone around the fungal hyphae indicated the ability of the fungus to solubilize inorganic phosphorous. Each treatment was replicated three times. The performance of each fungus was marked by assigning - and + sign. The - sign indicates no P solubilisation; + sign indicates small amount of P dissolved; ++ denotes medium amount of P dissolved; +++ indicates high amount of P dissolved. Percent solubilization efficiency and phosphate solubilization index was calculated as:

 

Where,
SI = Solubilization index.
SE = Solubilization efficiency.
Z = Clear zone diameter (mm).
R = Colony diameter (mm).
Identification of fungal isolates
 
Based on the morphological study, the fungal isolates were identified from ITCC and NFCCI, India (Table 1).
 

Table 1: Identification of fungal isolates from Indian Type Culture Collection (ITCC), New Delhi and National Fungal Culture Collection of India (NFCCI), Pune, India.


 
Promotion of growth of rice plant
 
Influence on growth of rice seedlings following treatment with different fungal isolates
 
Two isolates Trichoderma harzianum TaK12 and Trichoderma aureoviride TaN16 showed maximum shoot length and root length after 15 days of treatment with 20% maize grain inocula (MGI). In addition, they both showed maximum percent of seed germination after 15 days of treatment with 20% MGI. Maximum vigor index together with growth of rice seedling was found with the treatment of spore suspension of Trichoderma harzianum TaK12 after 15 days with 20% of MGI (Table 2 and 3).
 

Table 2: Effect on shoot length and root length of rice seeding following treatment with spore suspension (1´108 cfu ml-1) and maize granules inocula (MGI) of different fungal isolates.


 

Table 3: Effect on seed germination and vigour index of rice seedling following treatment with spore suspension (1´108 cfu ml-1) and maize granules inocula (MGI) of different fungal isolates.


 
Earlier reports support the findings of the current study. Doni et al., 2014 reported rice seeds treated with Trichoderma spp. SL2 improved germination rate along with seedling vigor (Doni et al., 2014). Co-inoculation of Trichoderma asperellum and Pseudomonas fluorescens were reported to promote fresh weight, shoot height, tiller numbers and dry biomass of rice and was found to be more effective as compared to single inoculation (Singh et al., 2020).
 
Indole acetic acid (IAA) production efficacy
 
Penicillium citrinum PcK10, Aspergillus niger AnK1 and Trichoderma aureoviride TaN16 exhibited high amount of IAA production. Trichoderma harzianum TaK12 showed medium amount of IAA production. Small amount of IAA production was shown by Talaromyces purpureogenus TpG11 (Fig 1, Table 4).
 

Fig 1: Production of Indole acetic acid (IAA) by fungal isolates collected from rhizosphere of rice of Uttar Dinajpur, West Bengal.


 

Table 4: In vitro indole acetic acid (IAA) production by different fungal isolates of rice rhizosphere of Uttar Dinajpur, West Bengal.


 
There are several studies that support the production of IAA by Trichoderma, Aspergillus, (Pedrero-Méndezet_al2021, Tiru 2021).
 
Phosphate solubilisation efficacy
 
Trichoderma aureoviride TaN16 and Trichoderma harzianum TaK12 showed highest efficiency in phosphate solubilization with solubilization index (SI) 1.3 and 1.27 respectively. The isolates of Aspergillus species exhibited moderate response in terms of phosphate solubilization activity (Fig 2, Table 5). Filamentous fungi such as Aspergillus niger, Penicillium and Talaromyces were reported to be highly efficient in rock phosphate solubilization (Yin et al., 2015; Nelofer et al., 2016). Trichoderma viride, Trichoderma longibrachiatum, Trichoderma asperellum and Trichoderma harzianum were found with phosphate solubilization capability (Hewedy et al., 2020). These reports are in conformity with the findings of our present study.
 

Fig 2: In vitro phosphate solubilisation ability of fungal isolates collected from rhizosphere of different rice cultivated fields of Uttar Dinajpur, West Bengal.


 

Table 5: Phosphate solubilization efficiency of different fungal isolates

Rhizosphere represents a complex ecosystem on earth (Sharma et al., 2021). Interaction of plant with rhizospheric fungi play a key role not only on boosting plant productivity rather influence the overall soil fertility (Htwe et al., 2019). To minimize the usage of traditional fertilizers, the exploration of the beneficial rhizospheric root colonizing fungi can be advantageous in sustainable management of rice. Intense study of these eco-friendly fungi of the rhizosphere is needed to improve agricultural productivity in sustainable ways.
Authors are thankful to Indian Type Culture Collection (ITCC), New Delhi and National Fungal Culture Collection of India (NFCCI), Pune for their assistance in identification of fungal isolates with accession numbers.
None.

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