ENHANCEMENT OF RICE AND WHEAT SEED GERMINATION AND SEEDLING VIGOR BY BIOCONTROL AGENT, Cladosporium cladosporioides

Received 09 March, 2020 Revised 18 April, 2020 Accepted 20 April, 2020 Online 30 April, 2019


INTRODUCTION
The Cladosporium cladosporioides is a widely distributed cosmopolitan and grey pigmented mold fungus. The Cladosporium is one of the extensively studied and the largest genera of hyphomycetes (Dugan et al., 2004). It has also been isolated as saprophytes as well as associated with plants (Torres et al., 2017;Saranya et al., 2013;Bensch et al., 2010) and insects (Habashy et al., 2016;Abdel-Baky, 2000;Abdel-Baky et al., 1998). Several lines of evidence suggest that C. cladosporioides is a potent entomopathogenic fungus and thus a potential candidate for biocontrol of insect pests (Islam et al., 2019;Habashy et al., 2016;Eken and Hayat, 2008). The biocontrol fungus, C. cladosporioides significantly improve the growth of tobacco seedlings in vitro when they were co-cultivated without physical contact (Paul and Park, 2013).
The benefits of seed treatment with microbial antagonists can extend well beyond plant establishment. For example, treatment of maize seed with Bacillus amyloliquefaciens and Microbacterium oleovorans reduced populations of Fusarium verticilliodes and associated mammalian-active mycotoxins fumonisin B1 and B2 in grain (Pereira et al., 2007(Pereira et al., , 2011. Microbial antagonists can provide plant protection in situations where no chemical treatments are available, such as oilseed rape seed delivery of Serratia plymuthica for suppression of pathogen Verticillium dahlia (Muller and Berg, 2008). In this case efficacy of biocontrol agent varied with seed treatment method with bio-priming and pelleting of seed providing better plant protection than film coating, indicating the importance of developing an optimal seed treatment process for the microbial agent used (O'Callaghan, 2016). The different species of biocontrol agent viz. Trichoderma have attracted attention because of their effectiveness against various plant pathogens (Harman et al., 2004). This biocontrol agent has shown impressive results against many phytopathogenic fungi including M. phaseolina (Aly et al., 2007). Several recent reports indicate that the biocontrol fungi enhances tolerance to abiotic stresses during plant growth (Bae et al., 2009;Yildirim et al., 2006), in part due to improved root growth, improvement in waterholding capacity of plants, or enhancement in nutrient uptake (i.e., potassium); whereas, in the absence of stress, plant growth may or may not be enhanced. The goal of this study was to evaluate the ability of by biocontrol agent, C. cladosporioides BOU1 to improve rice and wheat germination and seedling performance as well as seedling vigor in good-quality seeds. This report describes plant growth promoting activity of a C. Cladosporioides, strain BOU1 in rice and wheat seeds.

MATERIALS AND METHODS
The biocontrol agent, Cladosporium cladosporioides, (Davidiellaceae: Capnodiales) (Fresen.) de Vries, isolate BOU1 (GenBank accession number-MG654669) was used to evaluate the growth of rice and wheat plant by germination percentage, germination index, vigor index-I and vigor index-II (Table 1). The fungal isolate was obtained from the farmer's rice field in Gazipur, Bangladesh (Islam et al., 2019), which was originally isolated from the brown planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). The fungus was cultured on PDA (potato dextrose agar) medium. Conidia of C. cladosporioides were collected from 15day-old cultures (maintained at 25 ± 1ºC, 70 ± 10% RH, and L 12: D 12 photoperiod) and were suspended in water. The conidia were quantified using a hemocytometer and a light microscope.
The seeds of rice and wheat were obtained from BADC and BARI, respectively ( Table 1). The seeds were surface sterilized with 70% ethanol followed by 5% sodium hypochlorite and finally washed with sterilized distilled water before placing for germination assay. One hundred seeds of each plant species were selected for each treatment then soaked in the respective C. cladosporioides in a flask containing spore suspension (1 × 10 7 conidia/ml) for 30 min. There were 6 Petri dishes for each treatment. The seeds soaked in sterilized distilled water served as control. The treated seeds were incubated for 15 days in sterilized Petri dishes at 25 ± 2 °C fitted with filter paper and each Petri dish was irrigated with 10 ml sterilized water. The root and shoot length of the seedlings were measured by selecting fifteen seeds randomly for each treatment. Shoot length was measured from the base of the primary leaf to the base of the hypocotyls and root length was measured from the tip of the primary root to the base of hypocotyl, with all measurements expressed in cm. Shoot and root weight were examined using digital weight scales and expressed in gm. All measurements were done according to the method by Dash (2012).

Seed germination rate
Germination rate was the average of number of seeds that germinate over the 7 and 15 day periods. Seed germination of each species was tested as per ISTA (Anonymous, 1985) and germination percentage (GP) of each replication was worked out by using following formula:

Germination Index
Numbers of seedlings emerging daily were counted from day 0 until day 15 of planting. Thereafter, a Germination Index (GI) was calculated by using the following formula as proposed by AOSA (1983):

Seedling vigor index
Seedling vigor index (SVI) was examined after 15 days of incubation using the formula given by Abul-Baki and Anderson (1973) based on germination (%) and seedling length (cm) or seedling dry weight (gm):

Statistical analysis
Data were analyzed with a one-way ANOVA. The statistical analyses were performed using the Proc GLM procedure (SAS Studio 3.6, 2017). Means were separated using Least Significant Difference (LSD) test at 5% level of significance.

RESULTS AND DISCUSSION
In the current study, it was observed that all the seed treatments were significantly superior over control (untreated seeds). All the variables of germination and seedling vigor of rice and wheat were increased by the conidial suspension of C. cladosporioides BOU1 (Figure 1-4). The biocontrol agent, C. cladosporioides remarkably enhanced the germination and vigor index of rice and wheat indicating its growth promoting effects of plant. Significant differences were observed among the treatment of germination percentage (F = 4.65; df = 3, 23; P = 0.0126; Figure 1  Biological seed treatments for control of seed and seedling diseases offer the grower an alternative to chemical fungicides. While biological seed treatments can be highly effective, it must be recognized that they differ from chemical seed treatments by their utilization of living microorganism. Storage and application conditions are more critical than with chemical seed protestants, and differential reaction to hosts and environmental conditions may cause biological seed treatments to have a narrower spectrum of use than some chemicals. Conversely, some microbial agents applied as seed treatments are capable of colonizing rhizosphere, potentially providing benefits to the plant beyond the seedling emergence stage. The microbial inoculants applied as seed treatments deliver microorganisms directly to the plant rhizosphere-the narrow zone of soil that surrounds the roots where plants interact directly with microorganisms (Philippot et al., 2013). It is a zone of intense microbial activity, with growth of plants and microorganisms dependent on reciprocal provision of nutrients and a wide range of other compounds including plant growth regulators and antibiotics. Many beneficial microorganisms of agricultural importance are rhizosphere colonizing species, with ability to increase plant growth via a range of mechanisms (Babalola, 2010).  Application of synthetic insecticides to control insect pest in crop field has seriously deteriorated the environment as well as increased the resistance in notorious crop pests to the insecticides. Considering the high cost and the deleterious effects of insecticides, biological control of insects through beneficial microorganisms is an eco-friendly alternative for sustainable agriculture. The fungal isolate of C. cladosporioides BOU1 not only controls the target insect pest, but also promotes the seed germination and seedling vigor of rice and wheat. Our current study is that the fungal isolate BOU1 significantly improved the germination and seedling vigor of rice and wheat. Growth promotion of plants by an entomopathogenic fungus is highly advantageous for its practical use in crop production. The plant-growth-promoting fungus (PGPF), Talaromyces sp. significantly enhanced the growth of Brassica campestris seedlings and their resistance to Colletotrichum higginsianum (Yamagiwa et al., 2011). The inoculation of the rice seeds with Trichoderma spp. significantly increased rice seed germination rate, vigor index and speed of germination as compared with untreated control (Doni et al., 2014). The seed treatments were significantly superior over control (untreated seeds). The germination percent, plumule length, radical length and vigor index in case of lentil and Terminalia arjuna showed better performance for all these attribute with chemical treatments (Shukla et al., 2008;Cokkizgin and Cokkizgin, 2008). The information concerning the effect of soil application and seed treatment with Trichoderma species on seed germination shoot, root length dry root in case of chickpea was also reported earlier (Dubey et al., 2011;Shahid et al., 2011;Kumar et al., , 2015.